Q J Med 2004; 97: 211-218
QJM vol. 97 no. 4 (c) Association of Physicians 2004; all rights reserved.
Microsomal triglyceride transfer protein polymorphisms and lipoprotein levels in type 2 diabetes
From the Department of Diabetes and Endocrinology, Trinity College Dublin and The Adelaide and Meath Hospital, Dublin, Ireland
Received 14 October 2003 and in revised form 19 January 2004
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Background: Microsomal triglyceride transfer protein (MTP) regulates the assembly of chylomicrons in the intestine and very-low-density lipoprotein (VLDL) in the liver. Common polymorphisms have been described that do not affect lipoproteins in non-diabetic subjects. Their effect in diabetes has not been described in a Caucasian population.
Aim: To investigate the association of these three common polymorphisms with lipoproteins in type 2 diabetes.
Methods: Eighty-two patients consumed a high-fat test meal. Chylomicron and VLDL apoB48, apoB100, cholesterol, triglycerides and phospholipids were measured fasting, and at 4 and 6 h postprandially. MTP genotyping was performed by PCR-RFLP.
Results: Thirty-three subjects were heterozygous for the -493 G/T substitution. These patients had significantly lower LDL cholesterol (3.0 ± 0.2 vs. 3.5 ± 0.1 mmol/l, p < 0.02). In the postprandial period, they had higher levels of apoB48 in the VLDL fraction (4 h, 7.0 ± 1.4 vs. 2.9 ± 0.4 µg/ml plasma, p < 0.002; 6 h, 6.4 ± 1.0 vs. 3.5 ± 0.5 µg/ml plasma, p < 0.05). In the VLDL fraction there was significantly less cholesterol at 4 and 6 h (p < 0.05). The -400 A/T substitution gave very similar lipoprotein results, but there was significant linkage dysequilibrium between the two polymorphisms. No association was found between the -164 T/C polymorphism and either plasma lipids or the postprandial lipid profile. ApoE genotype was also examined, but did not influence the above results.
Discussion: The common -493 G/T MTP polymorphism is associated with changes in VLDL and LDL in Type 2 diabetic patients. The importance of the changes in apoB48-containing small particles requires further investigation. The significantly lower LDL cholesterol suggests that this polymorphism may confer protection against atherosclerosis in type 2 diabetes.
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Introduction |
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Cardiovascular disease is up to four times more common in diabetes, and up to 50% of patients admitted to the coronary care unit have diabetes or impaired glucose tolerance. Hypercholesterolaemia, and in particular raised levels of LDL cholesterol, are an important cardiovascular risk factor for both diabetic and non-diabetic patients. There is considerable alteration in the triglyceride-rich lipoproteins in the postprandial period in diabetes,1 and these particles have an important influence on LDL composition and size.2,3 We and others have demonstrated an increase in the intestinally-derived chylomicron particles in diabetes,3–7 and we have shown reduction in these levels following improved diabetic control.8 Recently, the role of postprandial lipoproteins in atherogenesis has become more apparent, and Mero et al.9 have demonstrated a relationship between postprandial apoB48 and the severity of coronary heart disease in type 2 diabetic patients. Microsomal triglyceride transfer protein (MTP) regulates the production of the chylomicron in the intestine, and VLDL in the liver, by influencing the assembly of the lipoprotein particle.10 ApoB48 defines the intestinally-derived particle, and apoB100 the hepatically-derived particle. In animal studies we have demonstrated an increase in intestinal MTP mRNA expression and MTP protein in diabetes11,12 and also in non-diabetic animals with insulin resistance.13 The increase in MTP mRNA was associated with an increase in chylomicron particle number, a potentially atherogenic alteration.14 In type 2 diabetes, the common -493 G/T MTP polymorphism has been associated with surrogate markers of non-alcoholic hepatic steatosis.15 The homozygous form of the MTP -493 polymorphism has been shown to be associated with a higher concentration of small LDL 3 in type 2 diabetic patients of Chinese origin, but there was no effect in the heterozygous subjects.16 In non-diabetic subjects heterozygous for the T allele, no changes in VLDL or LDL triglyceride or cholesterol have been found, whereas the few subjects homozygous for the T allele had decreased numbers of lipid-rich VLDL particles and significantly lower total cholesterol and LDL cholesterol.17 It has been suggested that the T allele may interact with visceral obesity and hyperinsulinaemia in non-diabetic subjects.18 Examination of fasting lipids in a healthy young Black male population demonstrated that the rare T/T genotype was associated with a higher mean level of apoB related lipids, suggesting racial differences.19 The only study to date to examine the influence of the -493 G/T polymorphism on the postprandial lipoproteins revealed that subjects homozygous for the T/T polymorphism showed an increase in apoB48 in the smallest triglyceride-rich lipoprotein fraction postprandially, without a difference in postprandial triglycerides or in fasting plasma or LDL cholesterol, although there was a trend towards lower cholesterol and LDL cholesterol in the T/T variant.20 Other polymorphisms in the promoter region of the MTP gene have been identified, including the MTP -164 T/C, and the MTP -400 A/T.17,21,22 Information on the -164 T/C and -400 A/T polymorphisms is scant, with only three studies to date.17,22,22 The study by Herrmann et al.21 failed to find any association between either of the polymorphisms and plasma lipid levels. More recently, Ledmyr et al.21 found a significant association between the rare homozygous -164 T/C genotype and plasma total cholesterol and LDL cholesterol. They suggested that this might be due to the almost complete allelic association between this polymorphism and the -493 G/T polymorphism. Since type 2 diabetes is associated with major disturbances in the triglyceride-rich lipoproteins, and since in animal studies diabetes is associated with an increase in MTP expression, our hypothesis is that diabetes may amplify the effect of MTP polymorphisms on the assembly of apoB-containing lipoproteins, and that an alteration in the postprandial lipoprotein composition would result in lower LDL. The aim of the study was to examine the effect of the common -493 G/T gene polymorphism, and also the MTP -400 A/T and -164 T/C, on postprandial triglyceride-rich lipoproteins and LDL in type 2 diabetes
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Patients
Patients were recruited from the hospital diabetes unit. Inclusion criteria were that the patients were Caucasian, and in stable diabetic control, treated with diet or oral therapy. Exclusion criteria included abnormal renal or hepatic function, familial hypercholesterolaemia, unstable thyroid function, or being on lipid-lowering therapy. Eighty-two patients were both suitable and agreed to take part in the study. Ethics Committee approval was obtained for the study, and all subjects gave informed consent.
Design
On the day of the study, fasting blood was collected and plasma separated for lipoprotein measurement. Cells were stored frozen for MTP polymorphism determination and apoE genotyping. Patients were given an 1100 kcal high-fat test meal,8 and blood samples were taken at 4 and 6 h postprandially. After separation of plasma, the following preservatives were added to prevent oxidation and degradation of apoB: PPACK (1 mM), PMSF (0.1 mM), sodium azide (0.02% w/v), aprotinin (0.05 TIU) and EDTA (0.1%).
Lipoprotein isolation
Lipoproteins were separated from 10 ml plasma by the method of Havel et al.23 Plasma was overlaid with a 1.006 g/ml density solution, and centrifuged at 20 000 rpm at 4°C for 30 min in a Beckman L7-55 ultracentrifuge using a fixed-angle rotor. Chylomicrons were carefully removed from the top of the tube with a stretched Pasteur pipette, and the volume was measured. The density of the infranate was then adjusted to 1.006 g/ml, and the solution was centrifuged at 40 000 rpm at 4°C for 18 h to isolate VLDL. Lipoprotein fractions were stored at 4°C and lipoproteins measured within one week.
Chylomicron and VLDL apolipoprotein B48 and B100 determination
Chylomicron and VLDL apolipoprotein B48, and apo B100 were separated by SDS-polyacrylamide gel electrophoresis using 4–15% gradient gels (Biorad) as previously described.8 Non-delipidated lipoprotein samples (40 µg of protein) were reduced and applied to 4–15% SDS-polyacrylamide gels. Following electrophoresis, gels were stained with Coomassie Brilliant Blue. An apoB100 standard was prepared from LDL (density 1.025–1.063 g/ml) from a single individual, and was stored at -20°C and used throughout the study. Since the chromogenicity of apoB48 has been shown to be similar to that of apoB100,24 the concentrations of both apoB48 and apoB100 could be determined from this standard. ApoB48 and apoB100 staining was linear within the range 0.1–20 µg of protein. The bands were quantified by densitometry using Vilber Lourmat equipment (Vilber Lourmat Biotechnology) and Bio1D v6.32 software (Vilber) for analysis. Density values were assigned to the apo B100 band of the human LDL and a standard curve constructed, with the values being recalculated by linear regression. Curves with a correlation coefficient > 0.95 were accepted. Results were expressed as µg/ml plasma. The intra-assay variations were 2.8% and 3.9% for apoB48 in the chylomicron and VLDL fractions and 4.8% and 6.8% for apo B100 in the two fractions. The inter-assay coefficient of variations were 5% and 7% for apoB48 and 8% and 8.6% for apoB100, in the chylomicron and VLDL fractions, respectively.
Biochemical analyses
Venous blood glucose levels were determined by an enzymic colorimetric method using a commercially available diagnostic kit (Boehringer Mannheim). Blood HbA1c was determined using an enzyme immunoassay kit, containing monoclonal antibody specific for HbA1c (Novo Nordisk). The normal value was taken as < 5.8%. Commercially available diagnostic kits were used to determine lipoprotein composition. Total cholesterol and triglyceride content of lipoprotein fractions were measured by an enzymic colorimetric method using kits from Boehringer Mannheim, and phospholipids were assayed using a kit from Biomérieux.
DNA analysis
Genomic DNA was isolated using the Puregene DNA isolation kit (Gentra Systems). The method for detecting the -400 and -493 G/T polymorphisms was adapted from the method of Karpe et al.17 A 109-bp DNA product, encompassing the -493 site, was generated by PCR with a 5' mismatched primer MTP493 1 (5'-GGATTTAAATTTAAACTGTTAATTCATATCAC) and a 3' primer MTP493 2 (5'-AGTTTCACACATCAAGGACAATCATCTA). The PCR was performed in 50 µl containing 0.2 mM of each dNTPs, 2.5 U Taq DNA polymerase, 40 pmol of each primer and 10x Taq DNA polymerase buffer (50 mM KCL (pH 8.3), 10 mM Tris-HCl and 1.5 mM MgCl2). The reactions were carried out on a DNA engine thermal cycler (MJ Research). After an initial denaturation step (3 min at 94°C) the reactions were incubated as follows: 94°C for 1 min, 47°C for 1 min and 72°C for 1 min for 35 cycles, followed by a final elongation step at 72°C for 5 min. PCR products were incubated overnight at 37°C with the restriction enzyme HphI (1U). Restriction fragments were then separated on 12% polyacrylamide and sized by comparison to a DNA step ladder Puc 18 (Promega). This restriction digestion gives rise to one full-length fragment of 109 bp for homozygotes for the T variant, two fragments of 89 and 20 bp for homozygotes for the G variant, and three fragments of 109, 89 and 20 bp for GT heterozygotes.
Primers MTP400 1 (5'-CCCTCTTAATCTCTTTCCTAGAA) and MTP400 2 (5'-aagaatcatattgaccagcaatc) were used for genotyping of the -400 A/T polymorphism. The MgCl2 concentration was increased to 2 mM and the PCR was further optimized by changing the procedure to 35 cycles at 94°C for 0.5 min, 55°C for 1 min and 72°C for 1 min, followed by a final elongation step of 72°C for 5 min. PCR products were then incubated overnight at 37°C with the restriction enzyme Ssp1 (4U). The -400 A allele gives rise to a full-length fragment (838 bp), whereas the -400 T allele gives rise to a cutting site, giving one full-length fragment of 838 bp for homozygotes for the A variant, two fragments of 494 and 344 bp for homozygotes for the T variant, and three fragments of 838, 494 and 344 bp for AT heterozygotes.
Genotyping of the MTP -164 T/C polymorphism was performed as previously described.15 The MgCl2 concentration was 2 mM. Primers used were MTP164 1 (5'-GGTTTGGTTTAGCTCTCAAAAGTG) and MTP164 2 (5'AGTGAGGGAGTGACC CTCTTC). The amplification cycle started with denaturation at 94°C for 3 min, followed by 40 cycles at 94°C for 45 s, 60°C for 30 s and 72°C for 1 min, followed by a final elongation step of 72°C for 5 min. PCR products were digested with Bsr1 (10U). This restriction digestion gives rise to one full-length fragment of 220 bp for homozygotes for the TT variant, two fragments of 176 and 44 bp for homozygotes for the CC variant and three fragments of 220, 176 and 44 bp for TC heterozygotes.
Genotyping for the ApoE polymorphism was performed using the Amplification Refractory Mutation System (ARMS) as previously described by Wenham et al.25
Statistics
Statistical analysis used the Student's t-test for comparison of fasting and postprandial levels in diabetic subjects. Non-parametric tests were used for unequal distributions. Results are expressed as the mean±SEM. Inter- and intra-assay variation is expressed as SD/mean x 100. A p value of < 0.05 was regarded as statistically significant. Allele frequencies were calculated by gene counting. The normalized linkage disequilibrium coefficients were calculated using Arlequin Software.
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Results |
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MTP -493 G/T polymorphism
Forty-five subjects (55%) were homozygous for the MTP -493 G allele, 33 (40%) were heterozygous G/T and four were homozygous for the T allele (5%). Patient details are given in Table 1. There was no difference in the sex ratio between the groups. Serum LDL cholesterol was significantly lower in subjects with the T allele (p < 0.05), and total cholesterol was reduced by 8% in carriers of the T allele, not quite reaching statistical significance (p < 0.058). In the chylomicron fraction (Table 2) there was no significant difference in apo B48 or apo B100 at any time point between the G/T and G/G subjects. In the VLDL fraction (Table 3) there was a significant increase in apo B48 at 4 h (p < 0.002) and 6 h (p < 0.05). There was a significant reduction in the cholesterol at 4 h and 6 h (p < 0.05) and a reduction in cholesterol/apo B at 4 h and 6 h (4.0 ± 0.8 vs. 2.4 ± 0.4 and 3.9 ± 0.9 vs. 2.7 ± 0.6, p < 0.05)
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MTP -400 A/T polymorphism
Patient characteristics and plasma lipids are shown in Table 4. Forty-one patients (50%) were homozygous for the A allele, 34 (40%) were heterozygous and eight (10%) were homozygous for the T allele. There was no difference in blood glucose, HbA1c, triglycerides or BMI between the groups. Plasma cholesterol was lower, and LDL cholesterol significantly lower, in the A/T group (p < 0.05) compared to the wild type A/A. In the chylomicron fraction, there was no significant difference between the MTP -400 A/T polymorphism groups in apo B48, apo B100, cholesterol or triglyceride at any time point. In the VLDL fraction, A/T polymorphism heterozygotes, when compared to wild type -400 A/A, showed increased apoB48, both fasting (3.9 ± 0.8 vs. 2.0 ± 0.4, p < 0.05) and at 4 h (7.4 ± 1.2 vs. 2.9 ± 0.4, p < 0.005) and 6 h (7.4 ± 1.2 vs. 3.5 ± 0.6, p < 0.005) with a tendency to lower apoB100 at all time points in carriers of the T allele. VLDL cholesterol was significantly lower fasting (57 ± 9 vs. 108 ± 15, p < 0.01) in heterozygous carriers of the T allele, and it was also lower at 4 h (88 ± 14 vs. 145 ± 14, p < 0.05) and at 6 h (88 ± 10 vs. 149 ± 17, p < 0.005) in that group. Eighty percent of the -400 A/T subjects also carried the -493 G/T polymorphism. All significant differences were obliterated when we reanalysed the few patients with -400 A/T polymorphism who did not also have the -493 G/T polymorphism.
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MTP -164 T/C polymorphism
No differences in fasting plasma lipids or fasting and postprandial chylomicrons and VLDL were observed in either heterozygous or homozygous carriers of the C allele.
Apo E genotype
ApoE genotype distribution among the -493 G/T and -400 A/T groups is shown in Table 5; 61% of subjects had apoE 3/3, 22% had apoE 3/4, 16% had apoE 3/2, and one patient had apoE 2/4. There was no significant difference in the distribution of the apoE genotype between each of the different MTP genotype groups. Examination of the influence of apoE genotype on the plasma lipids revealed the expected lower levels of LDL cholesterol in apoE 3/2 subjects (p < 0.02) and higher LDL cholesterol in the 3/4 subjects (p < 0.005). Blood glucose, HbA1c, triglycerides, HDL cholesterol and BMI were similar across groups. ApoE genotype did not significantly alter the postprandial profile. Exclusion of those who did not have the apoE 3/3 genotype did not change the results.
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Discussion |
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In this type 2 diabetic population, the heterozygous -493 G/T substitution (present in 40%) was associated with significantly lower LDL cholesterol and lower plasma cholesterol, suggesting that the effect of the T allele is magnified in diabetes. We did not have enough subjects to analyse the T/T variant separately, but have included the data for comparison. Ledmyr et al.22 examining 564 healthy White men in Sweden and 1117 healthy men from the WOSCOP study found that serum cholesterol was significantly lower in the rare T/T variant, and LDL cholesterol was also lower, compared to G/G subjects. Although these authors found that the T/T variant was associated with a significantly higher BMI and waist circumference, they found no difference in the common heterozygous form. St Pierre et al.18 have examined the role of the -493 G/T polymorphism in patients without diabetes, hypertension or CHD. They combined the homozygous and heterozygous T carriers and demonstrated that the T allele was associated with significantly lower cholesterol in subjects with low visceral adipose tissue or low fasting insulin, when compared to those subjects with high adipose tissue or insulin. They suggested that visceral obesity and hyperinsulinaemia modulate the effect of the MTP -493 G/T polymorphism. Our patients were obese, with no difference in BMI between the groups, and presumably insulin-resistant, although we did not measure insulin levels. It would certainly be interesting to repeat the study in lean diabetic patients. Chen et al.,16 examining Chinese patients, found that in type 2 diabetes the rare homozygous T/T was associated with higher concentrations of LDL3, whereas no effect was seen in controls. Although we did not subfractionate LDL or examine the composition, the lack of difference in serum triglyceride or in chylomicron or VLDL triglyceride suggests that LDL size may not have been different.
The postprandial phase is perhaps the most metabolically abnormal in diabetes,1 and we therefore hypothesized that the major affect of the T allele might be on the postprandial lipoproteins, and that this might lead to a reduction in LDL. In the Stockholm study, examination of a subset of 60 healthy 50-year-old White men, all heterozygous for the -493 T allele, did not reveal any significant alterations in the postprandial lipoproteins.26 In the postprandial state, there was however a significant increase in 3 h-postprandial apoB48 in the lowest density fraction in the T/T subjects, but no difference in the G/T group. In our study, examination of the postprandial phase showed a significant increase in apoB48 in the VLDL fraction. This was accompanied by a significant decrease at 4 h and 6 h in VLDL cholesterol content of these particles. Since VLDL is the precursor of LDL, these results would explain the significantly lower LDL in the G/T group.
The distribution of the -493 G/T MTP gene polymorphism in our patient population was similar to that found in France in diabetic patients, and in non-diabetic patients as part of the WOSCOPS study.15,22 The distributions of the -164 T/C and -400 A/T polymorphisms were also similar to those previously reported in a non-diabetic population.21,22 The rare allele frequencies for each of the polymorphisms were 0.27 for the -164 T/C, 0.33 for the -400 A/T and 0.25 for the -493 G/T, and these are similar to those found in a non-diabetic population.22 The -164 T/C, and to a greater extent the -400 A/T polymorphism, were in linkage disequilibrium with the -493 G/T polymorphism. The -400 A/T polymorphism had a normalized linkage disequilibrium coefficient (D') of 0.72 (p < 0.0001) and the -164 T/C polymorphism, a D' value of 0.27 (p < 0.0001). It is not surprising therefore, that we found similar lipoprotein alterations in the postprandial lipoproteins in the -400 A/T polymorphism. We had too few patients with the -400 polymorphism who did not also have the -493 G/T substitution to comment on the influence of the -400 A/T substitution in diabetes. Examination of subjects heterozygous for both polymorphisms compared to the -493 G/G group did not alter any results, suggesting that the -400 A/T polymorphism may be silent. The -164 T/C did not have any significant influence on the lipoprotein patterns in either the fasting or postprandial state in our patients, but the linkage disequilibrium with the -493 polymorphism was less pronounced.
ApoE is known to affect clearance of VLDL through a number of mechanisms. Carriers of the E2 allele have decreased VLDL clearance, leading to a higher VLDL and lower LDL cholesterol, whereas carriers of the E4 allele have accelerated clearance of VLDL with lower triglyceride and a higher LDL cholesterol. We therefore determined the apoE phenotype of the patients examined in this study. The apoE distribution was similar to that reported in the general Irish population.27 Exclusion of those who did not have the apoE 3/3 phenotype did not change the findings for the MTP polymorphisms.
We have previously shown an increase in MTP mRNA and protein levels in animals with diabetes.11,12 In the diabetic rabbit model, the MTP increase was associated with more small particles.12 We found more small intestinally-derived particles, also suggesting that the MTP -493 G/T polymorphism may up-regulate MTP activity. This is in agreement with the expression studies in hep G2 cells, which showed marked enhancement of transcription with the T variant.17 The present study also suggests that more small intestinally-derived particles are synthesized. The finding that the T allele in our study was associated with a lower LDL cholesterol may be explained by synthesis of the cholesterol-depleted VLDL particles that we found. Small VLDL particles form large buoyant LDL, which is cleared more quickly from the circulation: another explanation for the reduced LDL in our study.28
The postprandial lipoproteins are gaining favour in the aetiology of atherosclerosis, particularly since it has been shown that these particles can enter the sub-endothelial space, and because a specific apoB48 receptor has been identified on the macrophage,29 which may also help to attract these particles into the atherosclerotic plaque. Analysis of MTP genotype in large prospective studies is needed to discover whether the common -493 G/T polymorphism is associated with decreased risk of cardiovascular disease.
In conclusion, the changes in this study demonstrate that the effect of the -493 T allele is magnified in diabetes. The MTP -493 G/T polymorphism is associated with a decrease in the cholesterol content of the postprandial VLDL particles and lower LDL cholesterol.
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Acknowledgments |
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This study was funded by The Irish Heart Foundation, The Diabetes Federation of Ireland, and a generous donation from the Lynn Family in memory of the late Samuel Lynn. We are grateful to Professor Patrick Collins, Department of Biochemistry, Royal College of Surgeons in Ireland for providing laboratory facilities and for helpful criticism.
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Footnotes |
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Address correspondence to Professor G.H. Tomkin, 1 Fitzwilliam Square, Dublin 2, Ireland. e-mail gtomkin{at}rcsi.ie
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References |
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