majority of apo A-I and A-II are synthesized by the liver, as demonstrated by Ikewaki et al. [18]. In the
steady state, the fractional catabolic rate equals the fractional synthetic rate [19].
Because NIDDM patients were obese, the apoA-I and A-II production rates were calculated by multiply-
ing the apoA-I and A-II concentrations by the respec- tive FCR of these apolipoproteins. Thus, data were
normalized to the plasma volume of each subject.
2
.
5
. Analytical methods
2
.
5
.
1
. Cholesteryl ester transfer protein CETP
acti6ity and mass assays CETP activity in total human plasma was evaluated
by measuring the rate of transfer of radiolabeled cholesteryl esters CE from [
3
H]CE-HDL
3
toward the apoB-containing lipoprotein plasma fraction according
to the procedure previously described [20]. Briefly, mix- tures containing 25 ml of plasma, [
3
H]CE-HDL
3
2.5 nmol of cholesterol, and iodoacetate 75 nmol in a
final volume of 50 ml were incubated for 3 h at 37°C. Then, mixtures were ultracentrifuged at a density of
1.07 g ml
− 1
. The recovered d B 1.07 and d \ 1.07 frac- tions were mixed with scintillation fluid OptiPhase
Hisafe 3, Pharmacia, and radioactivity was counted for 5 min. Results were calculated as net percentages of
total radiolabeled CEs transferred from the density \ 1.07 g ml
− 1
to the density B 1.07 g ml
− 1
fractions, after deduction of the transfer occuring with control
sample incubated at 4°C. CETP mass concentrations were measured by using a
competitive enzyme-linked immunosorbent assay on a Biomek 2000 Beckman Instrument. CETP mass con-
centration values were determined in quadruplicate from a calibration curve obtained with a frozen plasma
standard [21].
Plasma glucose concentrations were measured by an enzymatic method glucose oxidase on a Vitros 750
analyzer Johnson Johnson Clinical Diagnostics, Rochester, NY. HbA1c was measured with ion ex-
change high performance liquid chromatography Bio- Rad, Richmond, CA. Serum insulin concentrations
were determined by radioimmunoassay CIS Bio Inter- national, Gif sur Yvette, France. Total and HDL
cholesterol, free cholesterol, triglyceride TG and apolipoprotein concentrations were measured in a
Cobas Fara analyzer Roche, Basel, Switzerland. Con- centrations of total, free and HDL cholesterol were
measured
enzymatically Boehringer
Mannheim, Mannheim, Germany, after a magnesium phospho-
tungstate precipitation for HDL cholesterol. Triglyce- rides were quantified with an enzymatic method
Roche. ApoB concentrations were measured by im- munoturbidimetry Boehringer Mannheim. ApoA-I
and apoA-II concentrations were determined by im- munoturbidimetry with anti apoA-I and A-II antibod-
ies purchased from Boehringer Mannheim. ApoA-I and apoA-II standards were purchased from Immuno AG
Vienna, Austria. CE was calculated as the difference between total and free cholesterol.
2
.
6
. Statistical analysis Results are expressed as means 9 SD. Statistical cal-
culations were performed using the SPSS software package. Control and diabetic groups were compared
using the non parametric Mann – Whitney U-test. Dia- betics before and after insulin therapy were compared
using the non parametric Wilcoxon matched-pairs test. Correlation coefficients were calculated by the Spear-
man test. P-values less than 0.05 were considered to be statistically significant.
3. Results
3
.
1
. Metabolic parameters Clinical and glucose metabolism characteristics are
presented in Table 1. The NIDDM patients were sig- nificantly overweight compared to controls BMI =
30.5 9 3.2 vs. 21.8 9 0.9 kg m
− 2
, P B 0.01 and did not change their weight on insulin therapy BMI = 30.6 9
3.5 vs. 30.5 9 3.2 kg m
− 2
. Insulin therapy significantly improved glycaemic control in NIDDM patients, as
assessed by fasting blood glucose concentration 8.16 9 1.11 vs. 11.93 9 1.89 mmol l
− 1
; P B 0.05 and HbA1c 7.8 9 1.4 vs. 9.3 9 1.5; P B 0.05. Insulin sensitivity
was evaluated in NIDDM patients by calculating the SSPG value during the insulin suppressive test. Two
month-insulin treatment did not significantly improve insulin sensitivity in our NIDDM patients SSPG value:
10.82 9 5.83 vs. 13.21 9 2.23 mmol l
− 1
normal value B
6.6 mmol l
− 1
.
3
.
2
. Plasma lipids At the beginning of the study, we checked in one
NIDDM patient that plasma triglyceride concentration was in steady state during the kinetic study. In this
patient, the coefficient of variation of triglyceridaemia calculated from plasma triglyceride concentrations mea-
sured throughout the kinetic study was 6 Fig. 1. Afterwards, in other subjects, plasma triglyceride con-
centration in the fed state was calculated as the mean value of 4 triglyceride measurements at time 0, 4, 8 and
12 h.
Before the introduction of insulin therapy, NIDDM patients presented high plasma triglyceride levels both
in the fasting and in the fed state, compared to control subjects 3.18 9 2.35 vs. 0.60 9 0.18 and 3.87 9 2.10 vs.
Fig. 1. Evolution of plasma triglyceride concentration in one NIDDM patient during the kinetic experiment. The coefficient of
variation for this parameter throughout the study was 6.
Fig. 2. Kinetic curves of VLDL apoB-100 obtained during a primed constant infusion of
L
-[1-
13
C]leucine. [
13
C] leucine enrichment values are expressed as percentage of plateau. Squares and triangles repre-
sent experimental values for one control subject and one NIDDM patient, respectively. The curves were obtained by monoexponential
modelling.
0.90 9 0.23 mmol l
− 1
, respectively; P B 0.01 Table 2. On insulin therapy, plasma triglycerides fell by 35 in
the fasting state 2.08 9 0.65 mmol l
− 1
, P B 0.05 and by 24 in the fed state 2.96 9 1,08 mmol l
− 1
, not significant. Nevertheless, plasma triglycerides remained
higher in NIDDM than in healthy subjects, both in the fasting and in the fed state P B 0.01. Total plasma
cholesterol concentration was initially comparable be- tween patients and controls, and was not modified by
insulin treatment in NIDDM patients. HDL cholesterol concentration was reduced in NIDDM patients before
insulin therapy 0.97 9 0.20 vs. 1.64 9 0.31 mmol l
− 1
and remained unchanged on insulin therapy 0.97 9 0.26 vs. 0.97 9 0.20 mmol l
− 1
. Before insulin treat- ment, CETP mass was not significantly different in
NIDDM patients and controls 2.12 9 0.46 vs. 2.49 9 0.19 mg l
− 1
, whereas CETP activity was increased, although not significantly, in NIDDM patients 33 9 13
vs. 25 9 3. Insulin therapy modified neither CETP activity in NIDDM patients 30 9 14 vs. 33 9 13, nor
CETP mass 2.05 9 0.63 vs. 2.12 9 0.46 mg l
− 1
. HDL particles were triglyceride-enriched in NIDDM
patients compared to control subjects, as assessed by the HDL TGprotein ratio 0.17 9 0.03 vs. 0.11 9 0.02,
P B 0.01 and by the HDL TGCE ratio 0.27 9 0.09 vs. 0.12 9 0.03, P B 0.01. Insulin therapy did not modify
these two ratios in NIDDM patients Table 2.
3
.
3
. ApoA-I and A-II kinetic parameters VLDL apoB-100 kinetic curves are shown in Fig. 2
for both a NIDDM patient and a control subject. HDL apoA-I and A-II kinetic curves are, respectively, shown
in Figs. 3 and 4 for a NIDDM patient before and after the introduction of insulin therapy, and for a control
subject. Absolute tracertracee ratio value at the plateau is not the same in two different subjects, despite
Table 2 Plasma lipid parameters of study subjects
a
NIDDM before insulin therapy Controls
NIDDM after insulin therapy n = 6
n = 5 n = 6
3.18 9 2.35 0.60 9 0.18
Fasting triglycerides mmol l
− 1
2.08 9 0.65
,
0.90 9 0.23 3.87 9 2.10
Triglycerides in the fed state mmol l
− 1
2.96 9 1.08 5.64 9 2.10
Total cholesterol mmol l
− 1
5.74 9 2.23 4.64 9 0.97
1.15 9 0.50
,
0.76 9 0.11 ApoB g l
− 1
1.22 9 0.62 HDL cholesterol mmol l
− 1
0.97 9 0.26 0.97 9 0.20
1.64 9 0.31 HDL TGprotein ratio
0.18 9 0.02 0.17 9 0.03
0.11 9 0.02 0.12 9 0.03
0.22 9 0.07 HDL TGCE ratio
0.27 9 0.09 2.05 9 0.63
CETP mass mg l
− 1
2.49 9 0.19 2.12 9 0.46
25 9 2 30 9 14
33 9 13 CETP activity transfer
a
Values are mean 9 SD. For the calculation of HDL TGprotein and HDL TGCE ratios, protein, TG and CE values are, respectively, expressed as g l
− 1
, mmol l
− 1
and mmol l
− 1
. PB0.05.
PB0.01 NIDDM before insulin therapy versus controls Mann–Whitney U test. PB0.05 NIDDM after versus before insulin therapy Wilcoxon matched pairs test.
PB0.05. PB0.01 NIDDM after insulin therapy versus controls Mann–Whitney U test.
Fig. 3. Kinetic curves of HDL apoA-I obtained during a primed constant infusion of
L
-[1-
13
C]leucine. [
13
C] leucine enrichment values are expressed as percentage of VLDL apoB-100 plateau. Symbols
represent experimental values in one control subject squares and in one NIDDM patient before closed triangles and after the introduc-
tion of insulin treatment open triangles. The curves were obtained by monoexponential modelling.
values as percentage of VLDL apoB-100 plateau which is presumed to represent apoA-I plateau.
Before the introduction of insulin therapy, HDL apoA-I concentration was decreased by 13 in
NIDDM patients compared to controls, but this differ- ence was not significant 1.16 9 0.12 vs. 1.33 9 0.14 g
l
− 1
Table 3. In NIDDM patients, HDL apoA-I FCR was accelerated by 70 0.39 9 0.11 vs. 0.23 9 0.01
pool d
− 1
, P B 0.01. This increase of HDL apoA-I FCR remained statistically significant after adjustment
for age. ApoA-I PR was increased by 45 0.45 9 0.12 vs. 0.31 9 0.04 g d
− 1
l
− 1
, P B 0.05. Before the intro- duction of insulin therapy, HDL apoA-II concentra-
tion, FCR and PR were similar in NIDDM patients and controls, even after adjustment for age 0.29 9 0.05
vs. 0.30 9 0.04 g l
− 1
; 0.23 9 0.06 vs. 0.19 9 0.02 pool d
− 1
; 0.065 9 0.019 vs. 0.056 9 0.009 g d
− 1
l
− 1
, respectively.
Insulin therapy did not induce any significant modifi- cation of HDL apoA-I and apoA-II concentration,
FCR and PR in NIDDM patients Table 3. HDL apoA-I FCR was significantly correlated with
apoA-I concentration r = − 0.47, P B 0.05, HDL cholesterol concentration r = − 0.68, P B 0.01, HDL
TGprotein ratio r = 0.62, P B 0.01, HDL TGCE ratio r = 0.54, P B 0.05, fasting TG 0.81, P B 0.001
and BMI r = 0.69, P B 0.01 Table 4.
4. Discussion
