the role of dietary cholesterol in the regulation of postprandial apolipoprotein b48 levels in...

ORIGINAL ARTICLES

The Role of Dietary Cholesterol in theRegulation of PostprandialApolipoprotein B48 Levels in DiabetesC. Taggart1, J. Gibney2, D. Owens1, P. Collins3, A. Johnson3, G. H. Tomkin*1,2

1Department of Clinical Medicine, Trinity College Dublin, Dublin,Ireland2The Adelaide Hospital, Dublin, Ireland3The Royal College of Surgeons in Ireland, Dublin, Ireland

The atherogenicity of intestinally derived postprandial lipoproteins has been confirmed ina number of recent studies. We have shown abnormalities in postprandial lipoproteinmetabolism in diabetic patients, a group with an increased susceptibility to atherosclerosis.This study examined the relationship between dietary cholesterol and the postprandial,intestinally derived, apolipoprotein B48 and apolipoprotein B100 from the liver. Wecompared 10 non-insulin-dependent (Type 2, NIDDM) diabetic patients and 10 age-matched non-diabetic control subjects. Fasting blood was taken and subjects were fed acholesterol-free, high fat meal. Blood samples were repeated at 2 h, 4 h, 6 h, and 8 hpostprandial. The following week fasting blood was collected and subjects were given thesame meal with 1g of added cholesterol. Blood was collected at the same timepoints. Chylomicrons and very low density lipoprotein were isolated by sequentialultracentrifugation and their lipoprotein composition determined. Apolipoproteins B48and B100 were separated by gradient gel electrophoresis and quantified by densitometricscanning using a low density lipoprotein apolipoprotein B100 standard. Post prandialchylomicron cholesterol and triglyceride increased after the high cholesterol meal in bothgroups (p , 0.001). The postprandial chylomicron apolipoprotein B48 response of bothdiabetic and control subjects to the cholesterol meal was less than to the cholesterol-freemeal (p , 0.001). Fasting very low density lipoprotein apolipoprotein B48 was higher indiabetic patients compared to control subjects and their postprandial increase followingthe cholesterol-free meal was significantly greater (p , 0.001). There was a 10-foldincrease in the incremental postprandial VLDL apolipoprotein B48 area under the curveafter the cholesterol-rich meal in the diabetic patients compared to a 3-fold increase incontrol subjects. The postprandial very low density lipoprotein apolipoprotein B100 wassimilar in the two groups with both meals. The study demonstrates a very significantincrease in the amount of intestinally derived small apolipoprotein B48-associated particlesin the very low density lipoprotein fraction following a cholesterol-rich meal in diabeticpatients. Synthesis rather than clearance may be the major cause of the increase in theseatherogenic postprandial particles. 1997 by John Wiley & Sons, Ltd.

Diabet. Med. 14: 1051–1058 (1997)

No of Figures: 3. No of Tables: 2. No of Refs: 39

KEY WORDS apolipoprotein B48; apolipoprotein B100; postprandial lipoproteins;chylomicron; very low density lipoprotein; non-insulin-dependent diabetesmellitus

Received 5 February 1997; revised 4 June 1997; accepted 2 July 1997

have previously shown that the non-insulin-dependentIntroduction(Type 2) diabetic (NIDDM) patient, who has a markedincrease in risk of developing atherosclerosis, has anAttention has been drawn to the possible atherogenicity

of post prandial lipoproteins.1–3 These are made up abnormal postprandial lipid metabolism with increasedtriglyceride-rich lipoprotein levels.4,5 Intestinal functionmainly of intestinally derived chylomicrons, chylomicron

remnants, and very low density lipoprotein (VLDL).4 We may be abnormal in diabetes in a number of respects.For example, in animals hypertrophy of the intestinalmucosa occurs and the rate of glucose absorption is

Abbreviations: VLDL very low density lipoprotein, apo B48 apolipoprot-increased.6,7 Altered intestinal absorption of sterols hasein B48, apo B100 apolipoprotein B100, apo E apolipoprotein E; AUC

area under curve, HbA1c haemoglobin A1c, PMSF phenyl methyl been demonstrated in diabetic patients8–10 and sterolsulphonyl fluoride, EDTA ethylenediaminetetra-acetic acid absorption has been shown to regulate intestinal choles-* Correspondence to: Professor G.H. Tomkin, 1, Fitzwilliam Square,

terol synthesis.8,11 Dietary cholesterol has been shownDublin 2, IrelandSponsors: The Adelaide Hospital Diabetic Research Fund to affect both intestinal and hepatic cholesterol synthesis

1051CCC 0742–3071/97/141051–08$17.50 1997 by John Wiley & Sons, Ltd. DIABETIC MEDICINE, 1997; 14: 1051–1058

ORIGINAL ARTICLESin man,12–14 but in the diabetic patient, the effect (cholesterol-free meal) and the following week the same

meal was repeated with 1g of added cholesterol (Merck,of dietary cholesterol on cholesterol homeostasis ispoorly understood. Darmstadt, Germany) dissolved in 10 ml of warm oil15

(cholesterol-rich meal). The sequence was not ran-The intestine has an important role in the assembly ofchylomicrons but little is known about the relevance of domized to ensure no carry-over effect of the cholesterol

to the second examination. The breakfast consisted of adisturbed intestinal function on chylomicron productionand metabolism. Cholesterol, whether absorbed from the standard serving of cereal (30 g) with skimmed milk

(75 ml), two slices of white bread (60 g) fried in adiet or synthesized in the intestinal mucosa, is transportedin the chylomicron which by definition is a particle commercial sunflower oil (Flora oil) (20 ml per slice), 1

grilled tomato, skimmed milk mixed with 10 ml ofcontaining one apolipoprotein B48 (apo B48) molecule.Apo B48, the structural protein for the chylomicron, is sunflower oil with or without cholesterol (to drink) and

a cup of tea or coffee (200 ml). The meal containednecessary for its assembly and therefore essential forintestinal absorption of dietary triglycerides and choles- 1300 kcal of which 55 % was fat and contained virtually

no cholesterol (cholesterol-free meal). Fasting bloodterol. Since apo B48 is produced exclusively in theintestine in humans, it is a useful indicator of intestinally samples were collected from each individual prior to

the meal and blood sampling was repeated at 2, 4, 6,derived lipoprotein particles.We have previously reported an increase in intestinally and 8 h after the meal. The breakdown of the fat content

was: saturated fat 12 %, polyunsaturated fat 63 %, andderived triglyceride-rich lipoproteins in NIDDM patientsusing an SDS page method for separating apo B48 and monounsaturated fat 25 %. This meal was chosen to

maximize lipoprotein changes and minimize changes inapo B100 isoforms.4,5,10 The aim of this study was toexplore the relationship between fasting and postprandial metabolic control in the diabetic patients. Egg yolk

cholesterol was not used as this would have increasedapo B48 in NIDDM and to examine the role of dietarycholesterol in the control of apo B48 and apolipoprotein the fat content of the test meal. Water, but no food, was

allowed during the study period. Patients and controlB100 (apo B100) in postprandial lipoproteins.subjects maintained their normal diets and medicationprior to, and during the study but sulphonylurea therapySubjectswas not taken on the day of each test.

Ten stable NIDDM patients attending the diabetic clinicwere asked to take part in the study. These were Lipoprotein Preparation and Analysiscompared with 10 non-diabetic control subjects of similarage, sex, and BMI selected from staff and patients Blood was centrifuged to separate plasma and cells and

the following preservatives added to prevent degradationattending the general medical outpatients clinics whohad been found to be free from disease. The patients’ of apo B: PPACK (1 mmol l−1), phenyl methyl sulphonyl

fluoride (PMSF) (0.1 mmol l−1), sodium azide (0.02 %),mean age was 63 ± 1.7 years and their mean BMI was28 ± 8 kg m−2. Their mean duration of diabetes was and ethylenediaminetetra-acetic acid (EDTA)

(0.1 mg ml−1). A chylomicron-rich sample was isolated6 ± 2.5 years. Diabetic patients were treated with dietalone (n = 3) or diet and sulphonylurea therapy (n = 7). by layering the plasma under an equal volume of saline

(density 1.006 g ml−1) containing the above preservativesNo patient was receiving insulin treatment. Patients withhepatic, renal or thyroid disease, and patients with any and centrifuging at 20 000 rev min−1 for 20 min at 4 °C.

Chylomicrons were washed and concentrated by re-gastric or intestinal symptoms, were excluded. No subjectwas taking drugs which would alter intestinal absorption layering under the same solution and re-isolated by

centrifugation for 20 min at 20 000 rev min−1 at 4 °C.or lipid metabolism. Control subjects had a mean ageof 64 ± 1.9 years and a mean BMI of 29 ± 1.2 kg m−2. VLDL (density ,1.006 g ml−1) was isolated from the

infranatant by ultracentrifugation at 40 000 rev min−1 forApolipoprotein E (apo E) genotype was determined forall subjects. Eight of the diabetic patients and seven of 24 h at 4 °C. Chylomicron and VLDL apo B48 and apo

B100 were analysed within 2 days and chylomicron andthe control subjects had apo E 3/3 genotype. Of theremaining control subjects, two has apo E 2/2 and one VLDL samples were stored at 4 °C and lipoprotein

composition measured within 1 week.had apo E 2/3 genotype. The remaining diabetic patientshad apo E 3/4. The study was approved by the Hospital Plasma, LDL, chylomicron, and VLDL cholesterol were

measured by enzymatic colorimetric methods using kitsEthics Committee and all subjects give informed consent.supplied by Boehringer Mannheim GmBH (Mannheim,Germany). Plasma, chylomicron and VLDL triglyceridesStudy Designwere measured with kits from BioMerieux (Charbonnieresles Bains, France). Blood glucose was measured bySubjects were given dietary advice prior to the study

and were instructed not to alter their diet between the an enzymatic method (Boehringer Mannheim GmBH,Mannheim, Germany) and haemoglobin A1c (HbA1c) wastwo meals. On two consecutive weeks, following an

overnight fast, subjects were given a test meal. On the determined using an enzyme immunoassay method(Novo Nordisk, Cambridge, England) (normal valuefirst occasion the meal contained virtually no cholesterol

1052 C. TAGGART ET AL.

Diabet. Med. 14: 1051–1058 (1997) 1997 by John Wiley & Sons, Ltd.

ORIGINAL ARTICLES,4.9 %). Serum total insulin was measured using a were made taking fasting value as zero. Results are

expressed as the mean ± standard deviation. Inter- andmicroparticle enzyme immunoassay (Abbott, IL, USA).Chylomicron, VLDL, and LDL protein was estimated by intra-assay variation is expressed as standard

deviation/mean × 100. A p value of ,0.05 was regardeda modification of the Lowry method.16

as statistically significant.Apolipoprotein E Genotyping

ResultsHuman genomic DNA was extracted from peripheralblood by a modification of the method of Adell and Clinical details for the subjects are shown in Table 1.

Diabetic patients were moderately controlled with aOgbonna.17 The apo E polymorphism of each subjectwas determined using the polymerase chain reaction mean HbA1c of 6.38 ± 0.78 % (normal value ,4.9 %).

Diabetic patients and control subjects had similar serumand digestion with the restriction enzyme Hha1 asdescribed by Hixson and Vernier.18 cholesterol and triglycerides.

Fasting plasma glucose was signifiantly higher in thediabetic patients and, as expected, there was a signifi-Apolipoprotein B Analysiscantly greater postprandial glucose excursion (p , 0.001,Figure 1). Fasting plasma insulin was similar in diabeticChylomicron and VLDL apolipoprotein B48 and apo

B100 were separated by SDS-polyacrylamide gel electro- and control subjects but the postprandial insulin responseto the high fat meal was significantly greater in the diabeticphoresis using 4–15 % gradient gels (Biorad, Herculas,

CA, USA) as previously described.4 Non-delipidated patients (p , 0.001, Figure 1). Addition of cholesterol tothe meal made no difference to the glucose or insulinlipoprotein samples (10 mg of protein) were reduced in

SDS sample buffer (3 % mercaptoethanol, 3 % SDS, response to food in either group.Fasting and postprandial chylomicron apo B48 concen-0.001 % bromophenol blue, 0.05 mmol l−1 Tris, 10 %

glycerol, pH 6.8) using a 2:1 ratio of sample to buffer trations for diabetic and control subjects after thecholesterol-free and cholesterol-rich meals are shown infor 4 min at 96 °C. Samples (10 ml) were applied to the

gel and run at 200 V until the front had reached the end Figure 2. The postprandial chylomicron apo B48 AUC,calculated using fasting value as zero, was significantlyof the gel (approx 40 min). Gels were stained for 1 h

with Coomassie Brilliant Blue (0.1 % in methanol:acetic greater with both meals in the diabetic patients comparedto control subjects (p , 0.001, Table 2). Following theacid:water 4.5:1:4.5) and destained with several changes

of the same solvent. Since the chromogenicity of apo cholesterol-rich meal, the postprandial chylomicron apoB48 (AUC, calculated using fasting value as zero) in theB48 has been shown to be similar to that of apo B100,19

a protein standard was prepared from LDL (density diabetic patients was 23 % lower than it had been withthe cholesterol-free meal (p , 0.001) and in the control1.019–1.063 g ml−1) of a single individual. LDL was

isolated by sequential ultracentrifugation20 and delipid- subjects it was 12 % lower than it was with thecholesterol-free meal (p , 0.001). Apo B48 concen-ated by ether extraction. The apolipoprotein purity of

the standard, tested by SDS-PAGE, was .99 % apo trations peaked at 6 h in control subjects but were stillrising in the diabetic patients following the cholesterol-B100. The standard was stored at −20 °C and used

throughout the study. Three concentrations (0.25 mg, rich meal.Fasting VLDL apo B48 levels were significantly higher in0.50 mg, and 0.75 mg) of this apo B100 preparation were

reduced in SDS sample buffer before being applied to diabetic patients compared to control subjects (p , 0.01,Figure 3). The response to the cholesterol-free mealall gels containing the postprandial TRL samples. Apo

B100 and apo B48 were linear within this range. (AUC, calculated using fasting value as zero) was notsignificantly different in the diabetic patients comparedApolipoproteins were quantified by densitometric scan-

ning using a Biorad densitometer. The inter- and intra- to control subjects (Table 2). There was a 10-fold increaseassay variations for apo B48 were 12.3 % and 6 %,respectively.

Table 1. Subject details

Statistical AnalysisDiabetic Controlsubjects subjects

Statistical analysis was performed using the unpairedStudent’s t-test for comparison of fasting levels in the n 10 10two groups, and by repeated measures of analysis of Serum triglyceride (mmol l−1) 1.97 ± 0.61 1.79 ± 0.58

Serum cholesterol (mmol l−1) 5.72 ± 0.55 5.70 ± 0.59variance (ANOVA), to examine the postprandial profileLDL cholesterol (mmol l−1) 3.91 ± 0.71 3.72 ± 0.38differences between the groups (minitab). Student’s pairedHDL cholesterol (mmol l−1) 1.19 ± 0.11 1.24 ± 0.09t-test was used to compare the cholesterol-free andHbA1C (%) 6.38 ± 0.78a 3.82 ± 0.3

cholesterol-rich meals within groups. Area under thecurve (AUC) measurements were obtained using the Mean ± SD.

ap , 0.01 with respect to control patients.formula – AUC = yo + 2y2 + 2y4 + 2y6 + y8. AUC analyses

1053DIETARY CHOLESTEROL AND APO B48 IN NIDDM

1997 by John Wiley & Sons, Ltd. Diabet. Med. 14: 1051–1058 (1997)

ORIGINAL ARTICLES

Figure 1. Plasma glucose and insulin postprandial profiles in NIDDM patients following a cholesterol-free s and a cholesterol-rich d high fat meal and control subjects following a cholesterol-free h and a cholesterol-rich j meal. Diabetic patients hadhigher fasting plasma glucose (p , 0.01) and an elevated postprandial response to a fat meal compared to non-diabetic subjects(P , 0.001). The two groups had similar fasting insulin levels but there was a significantly greater postprandial plamsa insulinexcursion in the diabetic patients (p , 0.001). There was no difference in the glucose or insulin response in either group whencholesterol was added to the meal

Figure 2. Chylomicron apo B48 postprandial profile in NIDDM and control subjects following a high fat, cholesterol-free s andcholesterol-rich d meal. Fasting chylomicron apo B48 was higher in NIDDM patients (p , 0.01). The postprandial incrementalapo B48 (AUC) was higher in NIDDM patients compared to control subjects. Following the cholesterol-rich meal, control subjectsshowed a similar postprandial apo B48 incremental increase to that for the cholesterol-free meal while the postprandial incrementalresponse in the NIDDM patients was significantly less with the cholesterol-rich meal compared to the cholesterol-free meal (p , 0.001)

in the diabetic patients’ VLDL apo B48 response when as zero) was similar in the diabetic patients andcontrol subjects after both meals. Chylomicron cholesterolcholesterol was added to the meal

(1.8 ± 0.7 vs 28.4 ± 1.2, p , 0.001). In control subjects, increased in both diabetic and control subjects followingthe cholesterol-rich meal (p , 0.001). Fasting VLDLthe postprandial increase (AUC) following the cholesterol-

rich meal was less than 3-fold greater than that for cholesterol was similar in all groups but the postprandialVLDL cholesterol response to both meals (AUC, calcu-the cholesterol-free meal (p , 0.001). There was no

difference in the fasting VLDL apo B100 in diabetic lated using fasting value as zero) was higher in thediabetic patients compared to control subjectspatients compared to control subjects (Table 2). With the

cholesterol-free meal the postprandial VLDL apo B100 (p , 0.001). Following the cholesterol-rich meal, thepostprandial cholesterol response decreased in diabeticAUC for the diabetic patients was significantly higher

than that of control subjects (p , 0.001). VLDL apo B100 patients (p , 0.001) while it increased in control subjects(p , 0.001). Fasting chylomicron triglyceride levels weredid not change significantly in either group after the

cholesterol-rich meal. similar in all groups. The postprandial response to bothmeals (AUC calculated using fasting value as zero) wasFasting chylomicron cholesterol was similar in the

four groups (Table 2). The postprandial chylomicron higher in the diabetic patients (p , 0.001) and bothdiabetic patients and control subjects increased theircholesterol response (AUC, calculated using fasting value



ORIGINAL ARTICLESTable 2. Postprandial chylomicron and VLDL composition (mg ml−1 plasma): comparison of cholesterol-free and cholesterol-rich meals

Diabetic subject Control subject

Cholesterol-free Cholesterol-rich Cholesterol-free Cholesterol-rich

Chylomicron apo B48fasting 18.1 ± 4.2a 16.0 ± 1.2 15.2 ± 1.6 16.2 ± 1.6AUC incremental 39 ± 2.6a 30 ± 2.1a,b 30.8 ± 1.3 26.6 ± 1.1b

VLDL apo B48fasting 3.0 ± 0.3a 4.3 ± 0.4a 1.8 ± 0.7 2.0 ± 0.2AUC incremental 3.8 ± 0.8a 33.0 ± 2.2a,b 4.1 ± 1.1 12.6 ± 1.7b

VLDL apo B100fasting 108 ± 8 105 ± 9 102 ± 6 104 ± 11AUC incremental 528 ± 15a 541 ± 11 511 ± 6 520 ± 10

Chylomicron cholesterol fastingAUC incremental 8.9 ± 1.6 10.2 ± 2.8 12.5 ± 49 12 ± 4

VLDL cholesterol 79 ± 6 101 ± 12b 81.5 ± 9 109 ± 8b

fastingAUC incremental 351 ± 51 375 ± 29 346 ± 26 344 ± 25

2011 ± 58a 1711 ± 71a,b 1374 ± 9 1601 ± 22b

Chylomicron triglyceridefasting 155 ± 46 126 ± 27 153 ± 29 151 ± 32AUC incremental 1721 ± 42a 2301 ± 54a,b 1298 ± 61 1634 ± 69b

VLDL triglyceridefasting 1100 ± 380 1680 ± 170 820 ± 30 1580 ± 425AUC incremental 13 800 ± 550a 29 800 ± 980a,b 7100 ± 200 13 700 ± 100b

Chylomicron cholesterol/apo B48VLDL cholesterol/apo B48 2.0 ± 0.2a 3.3 ± 0.6a,b 2.6 ± 0.2 4.1 ± 1.4b

AUC incremental 529 ± 7.0a 51.8 ± 4.6a,b 335 ± 3.2 127 ± 2.2b

Chylomicron triglyceride/apo B48AUC incremental 44 ± 7.0 76 ± 8.2a,b 42 ± 10.3 61 ± 13.2b

VLDL triglyceride/apo B48AUC incremental 3632 ± 70a 903 ± 9a,b 1731 ± 20 1087 ± 50b

Mean ± SD.ap , 0.001 different from control.bp , 0.001 different from corresponding group on cholesterol-free meal (paired t-test).

VLDL triglyceride response when cholesterol was added This may be relevant to the development of atherosclerosisin NIDDM patients. Almost 20 years ago Zilversmit1to the meal (p , 0.001).

The chylomicron cholesterol/apo B48 ratio (AUC/AUC) suggested that post-prandial remnant particles may beparticularly atherogenic and this has been confirmed.2,21–was higher in control subjects after both meals (p , 0.001)

and increased in both groups following cholesterol 23 The present study again demonstrates the increase inapo B48-containing particles in diabetes both fasting andfeeding (p , 0.001). The VLDL cholesterol/apo B48 ratio

was significantly higher in the diabetic patients compared postprandially. By using the cholesterol-enriched mealwe have demonstrated differences in apo B48 distributionto control subjects after the cholesterol-free meal

(p , 0.001) and decreased significantly in both groups in chylomicron and VLDL fractions between diabeticand non-diabetic subjects.after the cholesterol-rich meal (p , 0.001). Chylomicron

triglyceride/apo B48 increased in the two groups follow- Many studies have examined the effects of dietarylipid on postprandial lipoproteins in diabetic patientsing the cholesterol-rich meal (p , 0.001) while VLDL

triglyceride/apo B48 decreased in both groups and it has been suggested that cholesterol absorptionmay be decreased in NIDDM,8,24 the decreased absorp-(p , 0.001). Analyses of the above parameters were

examined excluding patients and controls who did not tion being related to the hypertriglyceridaemia andpossibly insulin resistance.8 A recent paper on non-have E3/E3 genotype. Although the means were slightly

different, re-analysis of the results did not alter levels diabetic subjects has shown a decrease in cholesterolabsorption in those patients with the highest bloodof significance.glucose.25 These patients had the highest cholesterolsynthesis. To our knowledge, no study has so farDiscussionexamined the effect of added cholesterol on chylomicronremnant particles.Our studies have suggested that increased intestinal

synthesis is responsible, at least in part, for an increase Our study suggests that increased postprandial choles-terol in the chylomicron fraction together with a decreasein apo B48-containing particles in diabetic patients.4,5,10



ORIGINAL ARTICLES

Figure 3. Apo B48 postprandial profile in the VLDL fraction in NIDDM and control subjects following a high fat, cholesterol-frees and cholesterol-rich d meal. Fasting Apo B48 levels were higher in diabetic patients compared to control subjects (p , 0.01).There was a 10-fold, postprandial incremental increase following the high cholesterol meal in diabetic patients (p , 0.001)compared to a nearly 3-fold increase in control subjects (p , 0.001)

in chylomicron apo B48 following the cholesterol- plasma cholesterol in the majority of subjects.14,27,28 Itis unlikely therefore that prolonged cholesterol feedingrich meal is associated with the cholesterol-enriched

chylomicron particle rather than a greater number of would generate different results. Prolonged feeding withdifferent types of dietary fat does alter postprandialchylomicrons. The important new finding in our study

was the very significant increase in the apo B48 in the lipoprotein metabolism in normal subjects29 but there isno suggestion that differing fatty acid diets will alterVLDL fraction in the diabetic patients, suggesting that

the NIDDM subject responds to a cholesterol enriched cholesterol absorption. Assessment of the dietary fat ofour diabetic and control subjects did not reveal anymeal by producing more apo B48 particles of smaller

size. Thus, the reduction in the chylomicron particles major differences either in quantity or quality. Dietarycholesterol, given in a bolus as in this study, is almostappears to be due to the synthesis of smaller intestinally

derived apo B48-containing particles which are now certainly trapped in the intestine for at least 24 h15 sothe cholesterol reaching the circulation has probablyfound in the VLDL and can no longer be called

chylomicrons. These particles have previously been been stored or newly synthesized in the intestine. It isinteresting that cholesterol feeding in the diabetic patientincluded with chylomicron remnants. Karpe et al.26 have

recently shown that the smaller apo B48-containing resulted in a slight, but significant, decrease in postpran-dial VLDL cholesterol. It is possible that the more rapidparticles in the VLDL fraction do not originate from large

chylomicrons and are probably directly synthesized by increase in chylomicron cholesterol in the diabeticsubjects inhibited hepatic cholesterol, but not apo B100,the intestine. Addition of cholesterol to a meal in

normolipidaemic non-diabetic subjects has been shown secretion. This would be supported by our previousfindings in animals, where diabetes was associated withto increase large TRL triglycerides and decrease plasma

esterified cholesterol.12 This is in keeping with our results. an increase in 3-hydroxy-3methylglutaryl co-enzyme A(HMGCoA) reductase levels in the intestine and aBecause cholesterol is very slowly absorbed, many

feeding studies have used a steady state study design. decrease in the liver.30

We were unable to find any previous studies on theThe present study was designed to examine the effect ofcholesterol loading in a single meal. Further studies effect of cholesterol-loading on postprandial apo B48 in

diabetes. However, we would have expected that if thewould be needed to examine whether a prolonged highcholesterol diet would increase or decrease the number problem was only related to clearance there would have

been an increase in chylomicron apo B48 particles, butof small chylomicron particles which are thought to beso atherogenic.2 Cholesterol metabolism is regulated by this was not the case. In fact, there was a decrease in

chylomicron apo B48 with an increase in the VLDLa precise feedback control mechanism and an acuteincrease in dietary cholesterol intake fails to increase fraction. These results strongly suggest an increase in



ORIGINAL ARTICLESsynthesis of abnormally small chylomicron particles. A containing particles in VLDL may be so atherogenic

since the percentage is not a measure of turnover.recent study in rats has demonstrated that small chylomic-This study suggests that the intestine may play a veryron particles are more slowly cleared.31 A situation

important part in cholesterol metabolism in the diabeticwhere there is an increase in small particle productionpatient. In diabetes the intestine appears to secrete moreand which leads to a delay in clearance of the particlesapo B48-containing particles and responds to dietarydue to their size is potentially atherogenic. Karpe etcholesterol by synthesizing smaller cholesterol-rich par-al.2 have demonstrated a relationship between smallticles which are found in the VLDL fraction. Thesechylomicron remnants and progression of coronaryintestinally derived particles have been shown to beatherosclerosis. Whether these particles are mainly for-associated with the progression of atherosclerosis in non-med as small particles or whether they become smalldiabetic subjects and if this is also the case in diabeticparticles once in the circulation is unkown but oursubjects then reduction in dietary cholesterol, with orresults would certainly support Karpe’s contention thatwithout drugs that inhibit cholesterol absorption, maythe majority of these particles are probably formed asbe particularly important in reducing atherosclerosis insmall particles and do not originate from the largethe diabetic patient.chylomicron particles.2,26 The suggestion that, in diabetes,

the intestine produces increased apo B48, is alsosupported by the significant decrease in the VLDL

Acknowledgementcholesterol/apo B48 in the diabetic patients followingthe same cholesterol load when compared to the control This study was supported by The Adelaide Hospitalsubjects. Microsomal triglyceride transfer protein (MTP) Diabetic Research Fund.transfers triglyceride between membranes and is essentialfor the assembly of apo B-containing lipoproteins. Haganet al.32 have demonstrated that the promoter activity of Referencesthe MTP gene is stimulated by cholesterol in humansand Wu et al.33 have shown a relationship in HepG2 1. Zilversmit DB. Atherogenesis: a post-prandial phenom-

enon. Circulation 1979; 60: 473–485.cells between the assembly of apo B-containing lipopro-2. Karpe F, Steiner G, Uffelman K, Olivercrona T, Hamsten A.teins and fatty acids, in particular oleic acid. These

Postprandial lipoproteins and the progression of coronarystudies may help to explain how the intestine regulates atherosclerosis. Atherosclerosis 1994; 106: 83–97.apo B-containing lipoprotein production. Finally Hagan 3. Meyer E, Westerveld HT, de Ruyter-Meijstek FG, Greeven-et al.32 demonstrated that insulin down-regulates the broek MJM, Rienks R, van Rijn HJM, et al. Abnormal

postprandial apolipoprotein B48 and triglyceride responsespromoter activity of the human MTP gene in thein normocholesterolaemic women with greater than 70 %transfected hamster model, again suggesting that instenotic coronary artery disease; a case–control study.

diabetes there would be an increase in assembly of Atherosclerosis 1996; 124: 221–235.apoB-containing lipoproteins. 4. Curtin A, Deegan P, Owens D, Collins P, Johnson A,

Tomkin GH. Intestinally-derived lipoprotein particles inAlthough gastric emptying is known to be delayednon-insulin-dependent diabetic patients with and withoutin hyperglycaemia and in patients with autonomichypertriglyceridaemia. Acta Diabetologica 1995; 32:neuropathy,34 it seems unlikely that our results could be 244–250.

explained on the basis since both the control subjects 5. Curtin A, Deegan P, Owens D, Collins P, Johnson A,and diabetic patients had similar incremental increases Tomkin GH. Alterations in apolipoprotein B-48 in the

post-prandial state in non-insulin-dependent diabetes.in apo B48 in the first 4 h.Diabetologia 1994; 37: 1259–1264.Apo E phenotypes have been shown to play an

6. Young NL, Saudek CD, Walters L, Lapeyrolerie J, Changimportant part in the regulation of cholesterol absorption V. Preventing hyperphagia normalises 3-hydroxy-3methyl-efficiency.35,36 We therefore re-analysed our data without glutaryl coenzyme A reductase activity in small intestine

and liver of diabetic rats. J Lipid Res 1982; 23: 831–838.the three diabetic patients and two control subjects who7. O’Meara N, Devery R, Owens D, Collins P, Johnson A,had apo E phenotypes other than apo E3/3. Repeated

Tomkin GH. Cholesterol metabolism in the alloxan-statistical analysis of the data did not yield different results. induced diabetic rabbit. Diabetes 1990; 39: 626–633.Karpe et al.2 investigated postprandial lipoproteins in 8. Sutherland WH, Scott RS, Linnott CJ, Robertson MC,

relation to coronary atherosclerosis. They found that the Stapely SA, Cox C. Plasma non-cholesterol sterols inpatients with non-insulin-dependent diabetes mellitus.postprandial plasma levels of small chylomicron remnantsHorm Metab Res 1992; 24: 172–175.related directly to the rate of progression of coronary

9. Briones ER, Steiger DL, Palumbo PJ, O’Fallon WM,atherosclerotic lesions. They found that the increment of Langworthy AL, Zimmerman BR, Kottke BA. Sterolplasma triglyceride during the postprandial period did excretion and cholesterol absorption in diabetics and

non-diabetics with and without hypercholesterolaemia.not reflect the amount of small chylomicron particles inAm J Clin Nutr 1986; 44: 353–361.the circulation or the progression of coronary lesions.

10. Curtin A, Deegan P, Owens D, Collins P, Johnson A,The preferential uptake of apo B48 by the liver23,37–39Tomkin GH. Elevated triglyceride-rich lipoproteins in

and the very rapid turnover of apo B48 may be the diabetes: a study of apo B48. Acta Diabetologia 1996;33: 205–210.reason why a relatively small percentage of apo B48-



ORIGINAL ARTICLES11. Farkkila MA, Tilvis RS, Miettinen TA. Cholesterol abosorp- 26. Karpe F, Bell M, Borkegren J, Hamsten A. Quantification

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the role of dietary cholesterol in the regulation of postprandial apolipoprotein b48 levels in...

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