Ross W. Milne

Molecular basis of lipid transfer protein deficiency in a family with increased high-density lipoproteins.

PLASMA high density lipoproteins (HDL) are a negative risk factor for atherosclerosis. Increased HDL is sometimes clustered in families, but a genetic basis has never been clearly documented1. The plasma cholesteryl ester transfer protein (CETP) catalyses the transfer of cholesteryl ester from HDL to other lipoproteins and therefore might influence HDL levels2. Using monoclonal antibodies, we show that CETP is absent in two Japanese siblings who have markedly increased and enlarged HDL. Furthermore, they are homozygous for a point mutation in the 5′-splice donor site of intron 14 of the gene for CETP, a change that is incompatible with normal splicing of pre-messenger RNA3. The results indicate that the family has an inherited deficiency of CETP due to a gene splicing defect, and illustrate the key role that CETP has in human HDL metabolism.

Increased prebeta-high density lipoprotein, apolipoprotein AI, and phospholipid in mice expressing the human phospholipid transfer protein and human apolipoprotein AI transgenes.

Human plasma phospholipid transfer protein (PLTP) circulates bound to high density lipoprotein (HDL) and mediates both net transfer and exchange of phospholipids between different lipoproteins. However, its overall function in lipoprotein metabolism is unknown. To assess the effects of increased plasma levels of PLTP, human PLTP transgenic mice were established using the human PLTP gene driven by its natural promoter. One line of PLTP transgenic mice with moderate expression of PLTP mRNA and protein was obtained. The order of human PLTP mRNA expression in tissues was: liver, kidney, brain, small intestine > lung > spleen > heart, adipose tissue. Western blotting using a human PLTP monoclonal antibody revealed authentic human PLTP (Mr 80 kD) in plasma. Plasma PLTP activity was increased by 29% in PLTP transgenic mice. However, plasma lipoprotein analysis, comparing PLTP transgenic mice to control littermates, revealed no significant changes in the plasma lipoprotein lipids or apolipoproteins. Since previous studies have shown that human cholesteryl ester transfer protein and lecithin:cholesterol acyltransferase only function optimally in human apoAI transgenic mice, the human PLTP transgenic mice were cross-bred with human apoAI transgenic mice. In the human apoAI transgenic background, PLTP expression resulted in increased PLTP activity (47%), HDL phospholipid (26%), cholesteryl ester (24%), free cholesterol (37%), and apoAI (22%). There was a major increase of apoAI in prebeta-HDL (56%) and a small increase in alpha-HDL (14%). The size distribution of HDL particles within alpha- and prebeta-migrating species was not changed. The results suggest that PLTP increases the influx of phospholipid and secondarily cholesterol into HDL, leading to an increase in potentially antiatherogenic prebeta-HDL particles.

Increased Levels of Apolipoprotein D in Cerebrospinal Fluid and Hippocampus of Alzheimer's Patients

Abstract: Apolipoprotein D (apoD) is a member of the lipocalin family of proteins. Most members of this family are transporters of small hydrophobic ligands, although in the case of apoD, neither its physiological function(s) nor its putative ligand(s) have been unequivocally identified. In humans, apoD is expressed in several tissues, including the CNS, and its synthesis is greatly increased during regeneration of rat peripheral nerves. As apoD may have an important function in the nervous system and, particularly, in nerve regeneration, we measured immunoreactive apoD levels in the hippocampus and in CSF of patients with either Alzheimers disease (AD) or other neuropathologies. In parallel, we determined the concentrations of apolipoprotein E (apoE), another apolipoprotein also implicated in nerve regeneration and in the etiology of AD. Levels of apoD but not apoE were increased in the hippocampus of AD patients compared with controls. ApoD concentrations, as determined by radioimmunoassay, were significantly increased in the CSF of AD patients (4.23 ± 1.58 µg/ml) and patients with other pathologies (3.29 ± 1.35 µg/ml) compared with those in the CSF of normal subjects (1.15 ± 0.71 µg/ml). Although the differences were smaller than for apoD, the mean apoE concentrations in the CSF of both groups of patients were also significantly higher than those of controls. In AD patients, apoD, but not apoE, levels in CSF and hippocampus increased as a function of inheritance of the ε4 apoE allele. This study therefore demonstrates that increased apoD levels in the hippocampus and in CSF are a marker of neuropathology, including that associated with AD, and are independent of apoE concentrations.

The intracellular redox state is a core determinant of mitochondrial fusion

Mitochondrial hyperfusion has recently been shown to function as a cellular stress response, providing transient protection against apoptosis and mitophagy. However, the mechanisms that mediate this response remain poorly understood. In this study, we demonstrate that oxidized glutathione (GSSG), the core cellular stress indicator, strongly induces mitochondrial fusion. Biochemical and functional experiments show that GSSG induces the generation of disulphide‐mediated mitofusin oligomers, in a process that also requires GTP hydrolysis. Our data outline the molecular events that prime the fusion machinery, providing new insights into the coupling of mitochondrial fusion with the cellular stress response.

Glycation of LDL by Methylglyoxal Increases Arterial Atherogenicity: A Possible Contributor to Increased Risk of Cardiovascular Disease in Diabetes

OBJECTIVE To study whether modification of LDL by methylglyoxal (MG), a potent arginine-directed glycating agent that is increased in diabetes, is associated with increased atherogenicity. RESEARCH DESIGN AND METHODS Human LDL was isolated and modified by MG in vitro to minimal extent (MGmin-LDL) as occurs in vivo. Atherogenic characteristics of MGmin-LDL were characterized: particle size, proteoglycan-binding, susceptibility to aggregation, LDL and non-LDL receptor–binding, and aortal deposition. The major site of modification of apolipoprotein B100 (apoB100) modification was investigated by mass spectrometric peptide mapping. RESULTS MGmin-LDL contained 1.6 molar equivalents of MG modification—mostly hydroimidazolone—as found in vivo. MGmin-LDL had decreased particle size, increased binding to proteoglycans, and increased aggregation in vitro. Cell culture studies showed that MGmin-LDL was bound by the LDL receptor but not by the scavenger receptor and had increased binding affinity for cell surface heparan sulfate–containing proteoglycan. Radiotracer studies in rats showed that MGmin-LDL had a similar fractional clearance rate in plasma to unmodified LDL but increased partitioning onto the aortal wall. Mass spectrometry peptide mapping identified arginine-18 as the hotspot site of apoB100 modification in MGmin-LDL. A computed structural model predicted that MG modification of apoB100 induces distortion, increasing exposure of the N-terminal proteoglycan–binding domain on the surface of LDL. This likely mediates particle remodeling and increases proteoglycan binding. CONCLUSIONS MG modification of LDL forms small, dense LDL with increased atherogenicity that provides a new route to atherogenic LDL and may explain the escalation of cardiovascular risk in diabetes and the cardioprotective effect of metformin.

Knockdown of Glyoxalase 1 Mimics Diabetic Nephropathy in Nondiabetic Mice

Differences in susceptibility to diabetic nephropathy (DN) between mouse strains with identical levels of hyperglycemia correlate with renal levels of oxidative stress, shown previously to play a central role in the pathogenesis of DN. Susceptibility to DN appears to be genetically determined, but the critical genes have not yet been identified. Overexpression of the enzyme glyoxalase 1 (Glo1), which prevents posttranslational modification of proteins by the glycolysis-derived α-oxoaldehyde, methylglyoxal (MG), prevents hyperglycemia-induced oxidative stress in cultured cells and model organisms. In this study, we show that in nondiabetic mice, knockdown of Glo1 increases to diabetic levels both MG modification of glomerular proteins and oxidative stress, causing alterations in kidney morphology indistinguishable from those caused by diabetes. We also show that in diabetic mice, Glo1 overexpression completely prevents diabetes-induced increases in MG modification of glomerular proteins, increased oxidative stress, and the development of diabetic kidney pathology, despite unchanged levels of diabetic hyperglycemia. Together, these data indicate that Glo1 activity regulates the sensitivity of the kidney to hyperglycemic-induced renal pathology and that alterations in the rate of MG detoxification are sufficient to determine the glycemic set point at which DN occurs.

Mapping of human apolipoprotein B antigenic determinants.

A minimum of 16 epitopes which provide a group of topographical markers to study the conformation of apolipoprotein (apo) B have been mapped in relation to elements of the sequence of apo B-100. Six of these epitopes are identified by monoclonal antibodies (Mabs) directed against low density lipoprotein (LDL) apo B, while at least 10 others react with Mabs obtained by immunization with delipidated and solubilized apo B. Five epitopes which are also expressed on apo B-48 have been assigned to the thrombolytic fragment T4 on the N-terminal side of apo B-100. None of these five epitopes requires the presence of lipids for its expression, suggesting that the conformation of the T4 region of apo B is more dependent on peptide-chain interactions than on peptide-lipid interactions. Four distinct epitopes have been assigned to the median thrombolytic fragment T3 of apo B-100, all of which require the presence of lipids for their expression; those epitopes closer to the C-teminus of T3 require specific interaction with cholesteryl esters. The same lipid dependence also characterizes a cluster of epitopes mapped to the N-terminal region of fragment T2. The epitopes that are close to the T2/T3 cleavage site and depend on the presence of cholesteryl esters for their expression are also those that react with the Mabs that inhibit the binding of LDL to its receptor. Therefore this region, which in addition contains two sequences with structural homology to the apo E receptor binding domain, probably constitutes a physiologically important receptor binding site for apo B. Finally, four other distinct epitopes which do not require the presence of lipids for their expression have been mapped on T2. In conclusion, the present report presents evidence that the immunochemical analogy of apo B-48 and apo B-100 is on the N-terminal half of apo B-100, whereas the apo B receptor binding domain is localized on the C-terminal half of apo B-100 close to the T2/T3 cleavage site.

Cholesteryl Ester Transfer Protein Directly Mediates Selective Uptake of High Density Lipoprotein Cholesteryl Esters by the Liver

Objective—To determine whether cholesteryl ester transfer protein (CETP) directly mediates selective uptake of high-density lipoprotein (HDL)-cholesteryl ester (CE) by hepatocytes and to quantify the effects of the CETP inhibitor, torcetrapib, on this process. Methods and Results—Using adenovirus-mediated CETP (ad-CETP) expression in primary mouse hepatocytes from either wild-type, low-density lipoprotein (LDL) receptor−/− or SR-BI−/− mice, we demonstrate that CETP enhances the selective accumulation of HDL-derived 3H-CE independently of known lipoprotein receptors. Addition of torcetrapib to the media did not impair the ability of cell-associated CETP to enhance CE uptake but reduced the ability of exogenously added CETP to increase selective uptake by up to 80%. When mice were infected with ad-CETP or ad-Luciferase and treated with daily intravenous injections of torcetrapib or vehicle, hepatic CETP expression resulted in a 50% decrease in HDL cholesterol in vehicle-treated animals versus a 33% decrease in HDL cholesterol in mice treated with torcetrapib. Conclusions—CETP mediates selective uptake of HDL-CE by hepatocytes by both torcetrapib-sensitive (exogenous CETP) and torcetrapib-insensitive (cell-associated CETP) mechanisms. Hepatic expression of CETP in vivo results in a marked decrease in cholesterol in particles in the HDL density range, consistent with a physiological role for hepatocyte CETP in selective uptake.

Modulation of apolipoprotein D and apolipoprotein E expression in rat hippocampus after entorhinal cortex lesion.

Apolipoprotein (apo) D is a member of the lipocalin family of proteins. Although its physiological function is unknown, apoD is thought to transport one or more small hydrophobic ligands. A second apolipoprotein, apoE is known to play an important role in lipid transport, and apoE genetic polymorphism has been shown to be associated with susceptibility to Alzheimers disease. Both apoD and apoE are expressed in the central nervous system (CNS) and both proteins accumulate at sites of peripheral nerve injury due to increased local synthesis. The two proteins may have overlapping or complementary functions within nervous tissue. In order to define the role of apoD within the CNS, we have studied the regional distribution of apoD and apoE mRNA and protein within the normal rat brain and the changes in apoD and apoE expression in the hippocampus of rats after entorhinal cortex lesion (EC lesion). Within the brains of normal rats, apoD expression in the hippocampus was as high as 180-fold that of the liver. ApoD mRNA levels in other areas of the rat brain ranged from 40 to 120 times the hepatic levels. The distribution of apoE gene expression within the brain was similar to that of apoD, but was much lower than hepatic apoE expression. When rats were subjected to EC lesion, the apoD message increased by 54% at 4 days post lesion (DPL) in the ipsilateral region of hippocampus while apoE mRNA levels (ipsilateral and contralateral) decreased by 43%. At 6 to 8 DPL apoD mRNA in the ipsilateral hippocampus remained elevated (42% above controls) whereas the apoE mRNA levels increased to about 15% above those of controls. At 14 and 31 DPL, both apoD and apoE expression was similar to controls. The increase in immunoreactive apoD in hippocampal extracts was more dramatic. At 1 DPL, immunoreactive apoD levels were already 16-fold higher than those in extracts of non-lesioned animals and, at 31 DPL, levels were still 8-fold higher than those of control animals. Finally, we have demonstrated that the levels of apoD in the brains of apoE-deficient mice are 50-fold those of wildtype control mice. ApoD clearly has an important function within the CNS in both normal and pathological situations.

Apolipoprotein D transcription occurs specifically in nonproliferating quiescent and senescent fibroblast cultures

We studied apolipoprotein D (apoD) mRNA in primary cultures of human diploid fibroblasts (HDF). In early‐passage HDF no apoD mRNA was detected in replicating cells in sparse culture, but the gene was expressed in quiescent cells in confluent and in serum‐starved cultures. In contrast, late‐passage HDF expressed apoD mRNA in sparse culture, but the level increased after attainment of confluence. Thus fibroblasts, the common cell‐type expressing apoD mRNA in vivo, express this characteristic following growth‐arrest. The same pattern of activation was found in another fibroblast cell line deficient in apoB/E (LDL) receptors, excluding a role for cellular cholesterol delivery by the LDL‐receptor pathway controlling apoD expression