Ian James Martins

Apolipoprotein E, cholesterol metabolism, diabetes, and the convergence of risk factors for Alzheimer's disease and cardiovascular disease

High fat diets and sedentary lifestyles are becoming major concerns for Western countries. They have led to a growing incidence of obesity, dyslipidemia, high blood pressure, and a condition known as the insulin-resistance syndrome or metabolic syndrome. These health conditions are well known to develop along with, or be precursors to atherosclerosis, cardiovascular disease, and diabetes. Recent studies have found that most of these disorders can also be linked to an increased risk of Alzheimers disease (AD). To complicate matters, possession of one or more apolipoprotein E ɛ4 (APOE ɛ4) alleles further increases the risk or severity of many of these conditions, including AD. ApoE has roles in cholesterol metabolism and Aβ clearance, both of which are thought to be significant in AD pathogenesis. The apparent inadequacies of ApoE ɛ4 in these roles may explain the increased risk of AD in subjects carrying one or more APOE ɛ4 alleles. This review describes some of the physiological and biochemical changes that the above conditions cause, and how they are related to the risk of AD. A diversity of topics is covered, including cholesterol metabolism, glucose regulation, diabetes, insulin, ApoE function, amyloid precursor protein metabolism, and in particular their relevance to AD. It can be seen that abnormal lipid, cholesterol and glucose metabolism are consistently indicated as central in the pathophysiology, and possibly the pathogenesis of AD. As diagnosis of mild cognitive impairment and early AD are becoming more reliable, and as evidence is accumulating that health conditions such as diabetes, obesity, and coronary artery disease are risk factors for AD, appropriate changes to diets and lifestyles will likely reduce AD risk, and also improve the prognosis for people already suffering from such conditions.

Cholesterol metabolism and transport in the pathogenesis of Alzheimer's disease

Alzheimer’s disease (AD) is the most common neurodegenerative disorder, affecting millions of people worldwide. Apart from age, the major risk factor identified so far for the sporadic form of AD is possession of the ɛ4 allele of apolipoprotein E (APOE), which is also a risk factor for coronary artery disease (CAD). Other apolipoproteins known to play an important role in CAD such as apolipoprotein B are now gaining attention for their role in AD as well. AD and CAD share other risk factors, such as altered cholesterol levels, particularly high levels of low density lipoproteins together with low levels of high density lipoproteins. Statins – drugs that have been used to lower cholesterol levels in CAD, have been shown to protect against AD, although the protective mechanism(s) involved are still under debate. Enzymatic production of the beta amyloid peptide, the peptide thought to play a major role in AD pathogenesis, is affected by membrane cholesterol levels. In addition, polymorphisms in several proteins and enzymes involved in cholesterol and lipoprotein transport and metabolism have been linked to risk of AD. Taken together, these findings provide strong evidence that changes in cholesterol metabolism are intimately involved in AD pathogenic processes. This paper reviews cholesterol metabolism and transport, as well as those aspects of cholesterol metabolism that have been linked with AD.

Bone mineral density, adiposity, and cognitive functions

Cognitive decline and dementia due to Alzheimers disease (AD) have been associated with genetic, lifestyle, and environmental factors. A number of potentially modifiable risk factors should be taken into account when preventive or ameliorative interventions targeting dementia and its preclinical stages are investigated. Bone mineral density (BMD) and body composition are two such potentially modifiable risk factors, and their association with cognitive decline was investigated in this study. 164 participants, aged 34–87 years old (62.78 ± 9.27), were recruited for this longitudinal study and underwent cognitive and clinical examinations at baseline and after 3 years. Blood samples were collected for apolipoprotein E (APOE) genotyping and dual energy x-ray absorptiometry (DXA) was conducted at the same day as cognitive assessment. Using hierarchical regression analysis, we found that BMD and lean body mass, as measured using DXA were significant predictors of episodic memory. Age, gender, APOE status, and premorbid IQ were controlled for. Specifically, the List A learning from California Verbal Learning Test was significantly associated with BMD and lean mass both at baseline and at follow up assessment. Our findings indicate that there is a significant association between BMD and lean body mass and episodic verbal learning. While the involvement of modifiable lifestyle factors in human cognitive function has been examined in different studies, there is a need for further research to understand the potential underlying mechanisms.

Intracellular Localization and Metabolism of Chylomicron Remnants in the Livers of Low Density Lipoprotein Receptor-deficient Mice and ApoE-deficient Mice EVIDENCE FOR SLOW METABOLISM VIA AN ALTERNATIVE apoE-DEPENDENT PATHWAY

The metabolism of chylomicron remnants in mice deficient in low density lipoprotein receptor (LDLr) or apolipoprotein E (apoE) was compared with that of control C57BL/6J mice. Mice were injected intravenously with chylomicron-like emulsions labeled with radioactive lipids. Blood samples were taken at fixed time intervals from the retro-orbital sinus, and clearance rates of the lipoproteins were assessed from the decline in plasma radioactivities. To follow the intracellular pathway of remnants in the liver, emulsions labeled with a fluorescent cholesteryl ester (BODIPY) were injected, and liver sections were processed and assayed by laser confocal microscopy. Catabolism of remnant cholesteryl esters was assessed by injecting emulsions labeled with cholesteryl[1-C]oleate and measuring the expired CO from each animal. In apoE-deficient mice, remnant removal from plasma was totally impeded, while the clearance of remnants in LDLr-deficient mice was similar to that in C57BL/6J control mice. The confocal micrographs of livers 20 min after injection of fluorescent chylomicron-like emulsions showed evenly distributed fluorescent particles in the hepatocytes from control mice. In contrast, the fluorescent particles were mainly located in sinusoidal spaces in LDLr-deficient mice. Three hours after injection the livers from control mice showed few fluorescent particles, indicating that remnants have been catabolized, while the sections from LDLr-deficient mice were still highly fluorescent. Micrographs from apoE-deficient mice showed no fluorescent particles in the liver at any time after injection. Measurement of expired radioactive CO after injection of emulsions labeled in the fatty acid moiety of cholesteryl oleate indicated that remnant metabolism was slower in the LDLr-deficient mice and essentially nil in the apoE-deficient mice. Control mice had expired 50% of the injected label by 3 h after injection. We conclude that under normal circumstances, chylomicron remnants are rapidly internalized by LDLr and catabolized in hepatocytes, with a critical requirement for apoE. When LDLr is absent, remnants are taken up by a second apoE-dependent pathway, first to the sinusoidal space of the liver, with subsequent slow endocytosis and slow catabolism. Hepatic clearance via this second pathway is increased by heparin, inhibited by lactoferrin, heparinase, and suramin, and down-regulated by feeding a high fat diet.

Lipid and apolipoprotein B48 transport in mesenteric lymph and the effect of hyperphagia on the clearance of chylomicron-like emulsions in insulin-deficient rats.

SummaryIn insulin-deficient streptozotocin-treated rats the intestine is hypertrophic and cholesterol synthesis and transport from the intestine are increased. The increased load of cholesterol is transported through the mesenteric lymph in chylomicrons. Clearance from plasma of injected chylomicrons is slowed in insulin-deficient rats, but the underlying mechanisms are currently unresolved. Hyperphagia may increase the size of chylomicrons which could contribute to defective chylomicron clearance in insulin-deficiency. In the present experiments we compared the size and number of chylomicrons in mesenteric lymph of control rats and diabetic rats infused with fat at two levels. In control and diabetic lymph-cannulated rats, as the infused dose of lipid increased the transport of triglyceride increased substantially compared with fasted rats. In contrast the transport of apoB48 increased by only a small amount during fat transport. Therefore, increased lipid transport was accomplished mostly by increased particle size, with only small increases in numbers of particles in intestinal lymph. Insulin-deficiency had no effect on triglyceride or apoB48 transport in lymph. Calculations suggested that each chylomicron particle contained a single molecule of apoB48. When hyperphagia in diabetic rats was prevented, the plasma triglycerides were decreased but the slow plasma clearance of injected chylomicron-like emulsions persisted. Hyperphagia, therefore, was unconnected to the impairment in chylomicron metabolism in insulin-deficient rats. Changes in the association with plasma apolipoproteins, in the expression of receptors for uptake of chylomicron remnants or in exposure to endothelial lipases may be responsible for the defective clearance of triacylglycerol-rich lipoproteins.

Apolipoprotein E influences amyloid-beta clearance from the murine periphery

The relationship between amyloid-beta protein (Abeta) metabolism and Alzheimers disease is currently poorly understood. While it is well known that the generation of Abeta results from enzymatic cleavage of its parent molecule, the amyloid beta protein precursor (AbetaPP), there is little information available regarding its in vivo clearance. The E4 isoform of apolipoprotein E (apoE) has been associated with poor clearance of Abeta under in vitro conditions. This is thought to be due to its poor ability to bind Abeta compared with the other common isoforms, apoE2 and apoE3. Although cell culture studies support the notion that Abeta clearance depends upon apoE isoform, validation of these findings requires Abeta clearance studies in vivo. In this study, we examined the clearance of Abeta in vivo from the periphery in mice that expressed apoE (C57BL/6J) or lacked apoE (APOE knockout). We measured the clearance of peripherally injected Abeta over time and additionally, the quantities sequestered by peripheral organs. Western blot analysis of the murine plasma indicated that the half-life of Abeta in the periphery was approximately 15 minutes. The livers of the C57BL/6J mice were found to have sequestered approximately 40% of the total injected Abeta at 90 minutes post-injection, whilst their kidneys contained 5% of the total injected Abeta. In contrast, the livers and kidneys of the APOE knockout animals were found to contain no detectable Abeta. These findings indicate that Abeta is rapidly removed from the plasma by murine peripheral tissues and the rate of its clearance is affected by apoE.

Effects of triacylglycerol-saturated acyl chains on the clearance of chylomicron-like emulsions from the plasma of the rat

We previously found that a single saturated acyl chain at the glycerol 2-position affected the metabolism of chylomicrons. The explanation for the effect is not clear, but could be reproduced by saturated monoacylglycerols. In the present work we have extended our measurements to several different triacylglycerols containing one or two saturated chains in specific locations in an attempt to define structural features that affect chylomicron clearance. Lipid emulsions containing triacylglycerol, egg yolk phosphatidylcholine, free cholesterol, cholesteryl oleate (CO) and labelled with 3H-CO and [14C]triolein (OOO) were prepared as models of lymph chylomicrons. When injected intravenously into rats, the metabolism of the emulsions was influenced by the acyl chains of the constituent triacylglycerols. Compared with emulsions containing OOO as the only triacylglycerol, plasma clearances of emulsion [3H]CO were extremely slow in emulsions containing either 1,2-dioleoyl-3-stearoylglycerol (OOS) or 1-stearoyl-2,3-dioleoylglycerol (SOO). As little as 10% of SOO in mixture with OOO slowed the clearance, and increasing proportions of SOO in OOO emulsions progressively slowed the removal of OOO and CO labels from plasma. With 50% and 100% SOO in the emulsions clearance was negligible. In emulsions containing the triacyl-sn-glycerols, 1,3-dimyristoyl-2-oleoylglycerol (MOM), 1,3-dipalmitoyl-2-oleoylglycerol (POP), 1-oleoyl-2,3-distearoylglycerol (OSS) or 1-palmitoyl-2-oleoyl-3-stearoylglycerol (POS), clearance rates of CO and OOO labels from plasma were significantly decreased compared with control OOO emulsions. With emulsions prepared with the triacylglycerols, 1-oleoyl-2,3-dimyristoylglycerol (OMM) and 1-oleoyl-2,3-dipalmitoylglycerol (OPP), clearances of CO label were significantly slower than with control OOO emulsions, while the removal of OOO label was not significantly affected. The uptake of CO label in the liver was decreased in conjunction with the lower rates of clearance of emulsion CO from the plasma. The clearance from plasma of 1,3-distearoyl-2-oleoylglycerol (SOS) emulsions was similar to the control OOO emulsions, but significantly more emulsion OOO label was taken up by the liver. Emulsions made with the triacylglycerols extracted from natural cocoa butter, which contained a high proportion of saturated acyl chains, were cleared similarly to the control OOO emulsions. Our findings indicate that the plasma clearance of triacylglycerol-rich lipoprotein particles depends upon the specific arrangements of the acyl chains of the constituent triacylglycerols, and not necessarily on the overall saturation of the triacylglycerols.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of phospholipid composition on the metabolism of triacylglycerol, cholesteryl ester and phosphatidylcholine from lipid emulsions injected intravenously in rats

Lipid emulsions were prepared with a similar size and lipid composition to natural lymph chylomicrons, but in which the surface phospholipid was either egg phosphatidylcholine, dioleoyl-, dimyristoyl-, dipalmitoyl- or 1-palmitoyl-2-oleoylphosphatidylcholine (EYPC, DOPC, DMPC, DPPC or POPC). When injected into the bloodstream of conscious rats, the emulsions containing EYPC or POPC were metabolized similarly to natural chylomicrons, consistent with rapid lipoprotein lipase-mediated hydrolysis of triacylglycerols, followed by hepatic uptake of the remnants derived from the emulsions. Phospholipids from the injected emulsions were removed more slowly and became associated with the high-density lipoprotein fractions of the plasma. Emulsions containing DPPC were metabolized differently. Triacylglycerols disappeared very slowly from plasma, indicating lack of hydrolysis by lipoprotein lipase, and phospholipid radioactivity did not transfer to high-density lipoprotein. With emulsions containing DMPC, the plasma removal rates for emulsion triacylglycerols and cholesteryl esters were fast, but phospholipid radioactivity failed to transfer to the high-density lipoprotein fractions of plasma. With DOPC emulsions, clearances were slower than EYPC or POPC emulsions, but transfer to high-density lipoproteins was efficient. Therefore, an unsaturated chain at the glycerol 2-position was necessary for rapid hydrolysis by lipoprotein lipase and for efficient transfer of phospholipids to high-density lipoproteins. With an unsaturated chain at the glycerol 2-position, a saturated chain at the glycerol 1-position optimized the rate of remnant removal from the plasma.

Evaluation of ion-exchange microspheres as carriers for the anticancer drug doxorubicin : in-vitro studies

Abstract— A comparison study of doxorubicin loading, release characteristics and stability within sodium and hydrogen forms of ion‐exchange resin microspheres has been performed. It was demonstrated that resins in the Na+ form, although having lower drug loading capacity, showed similar release profiles to resins in the H+ form but still maintain all the drug activity. Resins in the H+ form, despite having high drug loading capacity, caused drug degradation within microspheres due to their strong acidic nature. Therefore, in comparison with the H+ form, resins in the Na+ form can be considered as better carriers for doxorubicin in terms of sustaining the release of drug and maintaining drug activity. Other factors such as the degree of resin cross‐linkage and drug/resin mixing time have also been examined in relation to drug loading and release characteristics. Overall, this study demonstrated the significance of the characteristics of matrix materials and their influence on the drug activity and microsphere performance in‐vitro.

Chylomicron remnant metabolism studied with a new breath test in postmenopausal women with and without type 2 diabetes mellitus

objectives The kinetic basis for the effect of type 2 diabetes mellitus (DM) on postprandial lipoproteins has not been fully established. We investigated chylomicron remnant metabolism using a stable isotope breath test and fasting measurements of plasma apolipoprotein (apo) B‐48 and apoC‐III concentrations in postmenopausal women with and without type 2 DM.