D. Owens

Glycosylated low density lipoprotein is more sensitive to oxidation: implications for the diabetic patient?

Oxidised low density lipoprotein (LDL) is considered to be atherogenic. This study examined the relationship between glycosylation and oxidation of LDL from 10 normocholesterolaemic Type 2 diabetic patients, 10 hypercholesterolaemic Type 2 diabetic patients, and 10 normocholesterolaemic non-diabetic subjects. LDL was isolated by sequential ultracentrifugation and susceptibility to oxidation assessed by measuring thiobarbituric reactive substances (TBARS) during a 4-h oxidation period. LDL glycosylation was measured by aminophenylborate gel chromatography. Results demonstrated an increased susceptibility to oxidation in LDL from both diabetic groups, the mean 3-h TBARS values being 35.2 +/- 2.1 and 36.4 +/- 2.6 nmol MDA/mg LDL protein for normocholesterolaemic and hypercholesterolaemic diabetic patients compared with 24.5 +/- 2.5 nmol MDA/mg LDL protein for control subjects. LDL glycosylation of 2.20% +/- 0.11% and 2.89% +/- 0.46% for normocholesterolaemic and hypercholesterolaemic diabetic LDL was significantly higher than that for the non-diabetic control subjects of 1.60% +/- 0.12% (P < 0.02). There was a significant positive correlation (P < 0.005) between LDL glycosylation and LDL oxidation. The esterified/free cholesterol ratio which correlated positively with oxidation (P < 0.01) was significantly higher in LDL from both diabetic groups compared with LDL from control subjects (P < 0.01). Thus the increased incidence of atherosclerosis in diabetes may be related to glycosylation of LDL through its increased susceptibility to oxidation.

Messenger RNA levels of genes involved in dysregulation of postprandial lipoproteins in type 2 diabetes: the role of Niemann–Pick C1-like 1, ATP-binding cassette, transporters G5 and G8, and of microsomal triglyceride transfer protein

Aims/hypothesisThe aim of the present study was to examine the relationship between chylomicron composition and expression of genes that regulate chylomicron production in the intestine. We examined expression of the following: (1) Niemann–Pick C1-like 1 (NPC1L1), which regulates cholesterol absorption; (2) ATP-binding cassette transporters G5 and G8 (ABCG5, ABCG8), which regulate cholesterol homeostasis through their ability to excrete enterocyte cholesterol back into the lumen of the intestine; and (3) microsomal triglyceride transfer protein (MTTP), which packages the chylomicron particle by assembling cholesterol, triglyceride, phospholipids and apolipoprotein B48.Subjects, materials and methodsType 2 diabetic (26) and non-diabetic (21) patients were examined. Levels of NPC1L1, ABCG5 and ABCG8 and MTTP mRNA were measured in duodenal biopsies by real-time PCR. Lipoproteins were isolated by sequential ultracentrifugation.ResultsDiabetic patients had more NPC1L1 mRNA than the control subjects (p<0.02). Expression of ABCG5 and ABCG8 mRNA was lower in the diabetic patients (p<0.05) and MTTP expression was increased (p<0.05). There was a positive correlation between NPLC1L1 and MTTP mRNA (p<0.01) and a negative correlation between NPC1L1 and ABCG5 mRNA (p<0.001). Diabetic patients on statin therapy had increased ABCG5 and ABCG8 mRNA compared to those not on statin (p<0.02 and p<0.05) and less MTTP mRNA than those not on statin (p<0.05).Conclusions/interpretationThis study demonstrates that in type 2 diabetes there are important alterations to the expression of intestinal genes that regulate cholesterol absorption and chylomicron synthesis. In diabetic patients statin therapy is associated with reduced MTTP expression and increased ABCG5 and ABCG8 mRNA. The study suggests new mechanisms to explain postprandial diabetic dyslipidaemia and the beneficial effect of statins.

Elevated triglyceride-rich lipoproteins in diabetes. A study of apolipoprotein B-48.

The role of the intestine in cholesterol metabolism in human diabetes in unclear, although abnormalities have been demonstrated in cholesterol synthesis and absorption in diabetic animals. This study examines the relationship between fasting and post-prandial apolipoprotein B-48 in type 2 (non-insulin-dependent) diabetic and non-diabetic subjects. Eight type 2 diabetic patients and ten healthy non-diabetic control subjects were given a high-fat meal (1300 kcal), and the triglyceride-rich lipoprotein fraction was isolated by ultracentrifugation (d<1.006 g/ml) from fasting and post-prandial plasma. Apolipoprotein B-48 and apo B-100 were separated on 4%–15% gradient gels and quantified by densitometric scanning with reference to a purified low-density lipoprotein (LDL) apo B-100 preparation. Diabetic patients had significantly higher concentrations of apo B-48 and apo B-100 in both the fasting (P<0.05) and post-prandial (P<0.001) triglyceride-rich lipoprotein samples compared with non-diabetic subjects. The diabetic patients also exhibited a significantly different post-prandial profile for apo B-48 and apo B-100, with a prolonged increase and a later post-prandial peak, than the non-diabetic subjects (P<0.01). These results suggest that the raised fasting triglyceride-rich lipoproteins, often found in diabetes, are associated with apo B-48 and may be derived from increased intestinal chylomicron production. The post-prandial pattern suggests an abnormality in intestinal production as well as hepatic clearance of apo B-48 in type 2 diabetes.

Abnormalities in apo B-containing lipoproteins in diabetes and atherosclerosis.

Atherosclerosis is the major cause of death in patients with diabetes. Low‐density lipoprotein (LDL) being the most important cholesterol‐carrying lipoprotein has been studied extensively in both diabetes and non‐diabetes. This paper reviews the literature but also focuses on the precursors of LDL and in particular the postprandial apo B‐containing lipoproteins. Abnormalities in the postprandial lipoproteins and alteration in chylomicron assembly and clearance are discussed and the evidence presented suggesting the importance of dysregulation of these lipoproteins in atherosclerotic progression. The relationship between chylomicron production in the intestine and hepatic release of very low‐density lipoproteins (VLDL) is explored, as is the interrelationship between clearance rates of these lipoproteins. The size of LDL influences its atherogenicity. VLDL composition and size in relation to its influence on LDL is discussed. The effect of diet on the composition of lipoproteins and the relationship between fatty acid composition and clearance is reviewed. Evidence that diabetic control beneficially alters lipoprotein composition is presented suggesting how improved diabetic control may reduce atherosclerosis. The review concludes with a discussion on the effect of the apo B‐containing lipoproteins and their modification through glycation and oxidation on macrophage and endothelial function. Copyright

Oxidation of low-density lipoprotein in NIDDM: its relationship to fatty acid composition.

SummaryThe increased risk of atherosclerotic disease in diabetic subjects may be due to enhanced foam cell formation following an increased susceptibility of low density lipoprotein to oxidative modification. This study has compared fatty acid content and lipoprotein oxidisability in 10 non-insulin-dependent diabetic subjects with that in 10 control subjects. Both groups were normocholesterolaemic and the diabetic subjects had higher triglyceride levels (2.2±0.4 vs 1.2±0.2 mmol/l, p<0.05). The fatty acid composition was compared in low density lipoprotein following Folch extraction, separation by thin layer chromatography (for the lipid classes) and analysis by gas liquid chromatography. Low density lipoprotein oxidisability was assessed by conjugated diene and thiobarbituric acid reacting substance formation in the presence of copper ions. The esterified/free cholesterol ratio was higher in the low density lipoprotein from patients compared to control subjects (2.9±0.1 vs 1.9±0.3, p<0.05). Linoleic acid in the cholesteryl ester fraction of the lipoprotein was higher in the patients than in the control subjects (48.2±2.2% vs 42.4±3.4%, p<0.05) as was the total quantity of linoleic acid in the cholesteryl ester fraction (317.8±68.0 vs 213.2±28.0 Μg/mg protein, p<0.05) and in the low-density lipoprotein as a whole (443.2±70.0 vs 340.2±28.2 Μg/mg protein, p<0.05). Lipoprotein oxidisability was also increased in the diabetic group with increased formation of thiobarbituric acid reacting substances (35.6±7.2 vs 22.3±3.5 nmol/mg protein, p<0.05, increased total diene formation (502±60 vs 400±30 nmol/mg protein, p<0.05) and increased rate of diene formation (7.2±0.6 vs 5.1±0.9 nmol diene · mg protein−1 · min−1, p<0.05). This study indicates that low-density lipoprotein from diabetic subjects is more susceptible to oxidation. This could, in vivo, accelerate foam-cell formation thereby increasing atherosclerotic risk in diabetic subjects.

The Role of microsomal triglyceride transfer protein and dietary cholesterol in chylomicron production in diabetes

Aims/hypothesis. The aim of this study was to examine factors involved in chylomicron production in the streptozotocin diabetic rat, our hypothesis being that the synthesis of the chylomicron is abnormal in diabetes. Methods. Diabetic rats (n = 20) were paired with control rats (n = 20). Cholesterol emulsion was given by gavage and the lymph duct was cannulated. Lymph was collected for 4 h. Chylomicrons were prepared from the lymph by ultracentrifugation. Lymph apolipoprotein B48 was isolated by gradient gel electrophoresis and quantified by densitometric scanning. Intestinal microsomal triglycerol transfer protein mRNA was measured by solution hybridisation nuclease protection, using a rat specific [32P]-labelled cRNA probe. Results. Serum triglyceride and cholesterol were greatly increased in diabetic compared with control animals (258 ± 77 and 8.9 ± 6.4 mg/ml vs 1.04 ± 0.37 and 0.54 ± 0.03 mg/ml, p < 0.0001). Lymph chylomicron triglyceride and cholesterol were also higher in diabetic rats (29.4 ± 27.3 and 0.28 ± 0.3 mg/h vs 16.8 ± 10.6 and 0.18 ± 0.09 mg/h, p < 0.05). Lymph chylomicron apo B48 was similar in the two groups. Intestinal microsomal triglycerol transfer protein mRNA was higher in the diabetic rats (12.6 ± 3.2 vs 3.8 ± 3.0 amol/μg RNA, p < 0.0001) and there was a positive correlation between lymph triglyceride and microsomal triglycerol transfer protein mRNA in the whole group (r = 0.65, p < 0.01). Conclusion/interpretation. The study shows that microsomal triglycerol transfer protein mRNA is raised in diabetes without an increase in apolipoprotein B48 in the lymph suggesting that microsomal triglycerol transfer protein regulates chylomicron triglyceride content but not particle number. [Diabetologia (1999) 42: 944–948]

Microsomal triglyceride transfer protein: does insulin resistance play a role in the regulation of chylomicron assembly?

We have previously demonstrated that diabetes is associated with an increase in intestinal microsomal triglyceride transfer protein (MTP) mRNA in both the rat and rabbit models. The present study was designed to investigate the relationship between MTP expression and chylomicron assembly in an insulin resistant non-diabetic animal model. Ten insulin resistant Zucker obese fa/fa rats and ten lean fa/minus sign rats were examined at 8-10 weeks of age. The lymph duct was cannulated and lymph collected for 4 h. Lymph chylomicrons were isolated by ultracentrifugation and their composition determined. RNA was extracted from intestinal mucosa and from the liver. MTP mRNA was measured using the RNase protection assay. Blood sugar in the fatty rats was significantly higher (6.3 +/-1.2 vs. 5.4 +/-0.4 P<0.05) and plasma insulin was almost six times that of the lean rats (P<0.001). Plasma cholesterol and phospholipid but not triglyceride were significantly increased in the obese animals (P<0.01). Obese animals secreted significantly more lymph chylomicron apo B48 (0.05 +/-0.02 vs. 0.02 +/-0.01 mg/h P<0.005), triglyceride (9.7 +/-5.3 vs. 3.8+/-1.9 mg/h P<0.005) and phospholipid (1.5 +/-0.7 vs. 0.4 +/-0.3 mg/h P<0.001). The only difference in the chylomicron particle composition between the two groups was a significant increase in phospholipid (P<0.01). Intestinal MTP mRNA expression was significantly higher in the fatty compared to the lean rats (22.1 +/-9.5 vs. 7.8+/-5.6 amol MTP mRNA/microg total RNA P<0.001) as was hepatic MTP mRNA expression (6.9 +/-3.5 vs. 3.4 +/-1.5 amol MTP mRNA/microg total RNA, P<0.01). Thus in this animal model of insulin resistance, increased MTP, which was associated with increased chylomicron particle number, may play a crucial role in the development of atherosclerosis.

Cholesterol metabolism in alloxan-induced diabetic rabbits.

The effect of diabetes control on the activities of hydroxymethylglutaryl-CoA reductase (HMG-CoA reductase), cholesterol acyltransferase (ACAT), and phenol 2-monooxygenase, the major enzymes regulating cholesterol metabolism, was determined in alloxan-induced diabetic rabbits, and the results obtained were correlated with lipid and lipoprotein levels. Although intestinal HMG-CoA reductase activity was significantly increased (P < 0.001) in poorly controlled compared with moderately controlled diabetic rabbits, there was a significant reduction in the activities of intestinal ACAT (P < 0.01), hepatic HMG-CoA reductase (P < 0.05) and ACAT (P < 0.001), and phenol 2-monooxygenase (P < 0.01). The poorly controlled animals were hypercholesterolemic (P < 0.01), and this was reflected in the very-low-density and high-density lipoprotein fractions. Serum cholesterol levels in the nondiabetic and moderately controlled diabetic groups were similar. This increase in intestinal HMG-CoA reductase activity in the poorly controlled diabetic animals occurred in the absence of hyperphagia. Although abnormalities in cellular cholesterol metabolism could be partly responsible for the alterations in serum cholesterol levels in diabetes, the precise mechanisms underlying these enzymatic changes have yet to be elucidated.

Serum lipoproteins and cholesterol metabolism in two hypercholesterolaemic rabbit models

SummarySerum lipoproteins and key hepatic and intestinal enzymes regulating cholesterol synthesis, esterification and catabolism, namely 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase, acyl coenzyme A: cholesterol-o-acyltransferase (ACAT) and cholesterol 7α-hydroxylase respectively, were compared in two hypercholesterolaemic rabbit models — the cholesterol-fed animal and the hypercholesterolaemic diabetic animal. Hypercholesterolaemia in the cholesterol-fed animals was reflected in the VLDL and LDL fractions, whereas VLDL and HDL2 cholesterol levels were elevated in the diabetic animals. The lipoproteins of the cholesterol-fed animals were enriched with cholesterol but the lipoprotein fractions in the diabetic animals were enriched with triacylglycerol. While hepatic HMGCoA reductase activity was significantly reduced in both groups, the activities of hepatic ACAT and cholesterol 7α-hydroxylase were significantly increased in the cholesterol-fed animals and significantly reduced in the diabetic animals compared with controls. In the intestine, the activity of HMGCoA reductase was increased and ACAT reduced in the diabetic animals. By contrast, in the cholesterol-fed group, HMGCoA reductase activity was lower and ACAT activity was higher in comparison with the control group. These differences in lipoproteins and cellular cholesterol metabolism between the hypercholesterolaemic rabbit models may explain the differences in susceptibility to atherosclerosis, previously reported in these two animal models.

Autoantibodies to oxidized low density lipoprotein : the relationship to low density lipoprotein fatty acid composition in diabetes

Autoantibodies to oxidized low density lipoprotein have been shown to be an independent predictor of the progression of carotid atherosclerosis. This study examines the relationship between low density lipoprotein fatty acid composition and autoantibodies to both malondialdehyde‐modified and copper‐oxidized low density lipoprotein in non‐diabetic patients with (n  = 17), and without (n  = 18), definite evidence of previous myocardial infarction. The third group were non‐insulin‐dependent diabetic patients with no evidence of atherosclerosis (n  = 15) and the fourth group were patients with non‐insulin‐dependent diabetes (n  = 17) who had definite evidence of previous myocardial infarction. Fatty acids were measured by gas‐liquid chromatography. Antibodies to malondialdehyde‐modified low density lipoprotein and copper‐oxidized low density lipoprotein were determined by an ELISA method. Autoantibodies to copper‐oxidized low density lipoprotein were significantly higher in the non‐diabetic patients with heart disease when compared to any other group (p  < 0.05). Autoantibodies to malondialdehyde‐modified low density lipoprotein were significantly higher in the non‐diabetic subjects with heart disease and in both diabetic groups compared to non‐diabetic subjects without coronary heart disease (p  < 0.05). Lineolic acid (%) in low density lipoprotein did not differ between groups but arachidonic acid (%) was significantly lower in both diabetic and non‐diabetic patients with coronary heart disease (p  < 0.05). The diabetic patients with low antibodies had 39.6 ± 2.2 % polyunsaturated fatty acids in their low density lipoprotein while diabetic patients with high antibodies had 46.7 ± 1.2 % polyunsaturates in their low density lipoprotein (p  < 0.01). This study confirms the association between antibodies to oxidized low density lipoprotein and coronary heart disease and shows raised low density lipoprotein antibody levels in diabetic patients with and without demonstrable atherosclerosis. In the diabetic patients, those with high antibody levels had high polyunsaturated fatty acid levels in their LDL suggesting a possible role for dietary intervention.