NIDDM patients with poor metabolic control de- spite diet and antidiabetic oral therapy are often treated
by insulin. Insulin therapy generally improves gly- caemic control. Moreover it induces an important de-
crease of
hypertriglyceridaemia and
has been
demonstrated to make apoB-containing lipoprotein metabolism more physiological [8]. Unlike hypertriglyc-
eridaemia, HDL cholesterol level is moderately im- proved, or not improved at all on insulin therapy
[9 – 11]. The reasons for the persistence of low HDL cholesterol level on insulin therapy are not elucidated.
To our knowledge the metabolism of HDL particles has not been investigated in insulin-treated NIDDM
patients. The lack of modification of HDL cholesterol and apo A-I plasma concentrations may indicate that
the metabolism of HDL particles is unchanged by insulin therapy, but also that insulin may induce oppo-
site modifications on production and catabolism of these particles.
In order to get further insight into the persistence of low HDL cholesterol concentration in insulin-treated
NIDDM patients, we compared the kinetic of apolipo- proteins A-I and A-II, the two main apolipoproteins of
HDL particles, in NIDDM patients before and 2 months after the introduction of insulin therapy.
ApoA-I and A-II were endogenously labelled with
L
-[1-
13
C] leucine, a stable isotope, and sample isotopic en- richment
was quantified
by gas
chromatographycombustionisotope ratio mass spec- trometry, the most precise and most accurate technique
known at present [12]. In this article, we report that insulin therapy is unable to correct apoA-I hyper-
catabolism in NIDDM patients, and by analyzing the evolution on insulin therapy of factors likely to be
involved in apoA-I hypercatabolism, we elucidated the reasons for its persistence in insulin-treated NIDDM
patients.
2. Subjects and methods
2
.
1
. Subjects Six NIDDM patients and five healthy normolipi-
daemic subjects with normal glucose tolerance were studied. All subjects underwent physical examination
and laboratory tests for exclusion of hepatic, renal and thyroid abnormalities. Control subjects did not take
any medication. At their entry in the study, NIDDM patients were treated by oral antidiabetic therapy both
sulfonylureas glibenclamide 15 mg day
− 1
and met- formin 2550 mg day
− 1
in all patients. They were not taking any drug that could affect lipid metabolism.
Insulin treatment was introduced in NIDDM patients after the first kinetic experiment. It consisted in 2 daily
injections of intermediate acting human insulin at a dose of 0.26 – 1 U kg
− 1
day
− 1
. The patients performed three times daily capillary glucose monitoring. They
were educated in order to adapt insulin doses according to capillary blood glucose level. The goal was to obtain
a fasting glycaemic level between 4.5 and 8.25 mmol l
− 1
. The patients were asked to increase, by two units, their insulin dose when fasting glycaemia was above
8.25 mmol l
− 1
and to reduce it by two units, when fasting glycaemia was below 4.5 mmol l
− 1
. Physical and
biochemical characteristics
of controls
and NIDDM subjects are shown in Table 1. The experimen-
tal protocol was approved by the ethics committee of the Dijon University Hospital, and written informed
consent was obtained from each subject before the study.
2
.
2
. Experimental protocol Two kinetic studies were performed in each NIDDM
patient: the first before the introduction of insulin therapy and the second 2 months later.
Table 1 Clinical and glucose metabolism characteristics of study subjects
a
NIDDM before insulin therapy n = 6 NIDDM after insulin therapy n = 6
Controls n = 5 Age years
52.8 9 9.7 22.4 9 1.1
52.8 9 9.7 32
Sex malefemale 42
42 87.8 9 10.0
87.7 9 11.6 Weight kg
65 9 9.0 30.5 9 3.2
BMI kg m
− 2
30.6 9 3.5 21.8 9 0.9
Fasting blood glucose mmol l
− 1
11.93 9 1.89 8.16 9 1.11
,
4.38 9 0.28 7.8 9 1.4
9.3 9 1.5 HbA1c
6.18 9 1.15 10.83 9 7.63
Fasting insulin mUI l
− 1
13.21 9 2.33 SSPG mmol l
− 1
10.82 9 5.83
a
Values are mean 9 SD. 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.
The day before the kinetic experiment, insulin resis- tance was estimated in NIDDM subjects by the insulin
suppressive test, as explained in Ref. [13]. Shortly, insulin-stimulated glucose uptake was estimated by
measuring the steady state plasma glucose SSPG con- centrations achieved during the last 60 min of a 180-
min continuous infusion of somatostatin, insulin and glucose. Somatostatin, in this test, is used to suppress
endogenous insulin production, and insulin and glucose are infused at a dose of 0.8 mU kg
− 1
min
− 1
and 6 mg kg
− 1
min
− 1
, respectively. Normal subjects have SSPG below 6.6 mmol l
− 1
[13]. The kinetic study was performed in the fed state.
Food intake, with a leucine poor diet 1700 kcal day
− 1
, 55 carbohydrates, 39 fats and 7 proteins, was
fractionated in small portions which were provided every 2 h, starting 6 h prior to the tracer infusion, up to
the end of the study, in order to avoid important variations in apolipoprotein plasma concentration, as
previously performed by other groups [8,14]. The en- dogenous labelling of apolipoproteins A-I, A-II and
B-100 was carried out by administration of
L
-[1-
13
C] leucine 99 atom; Eurisotop, Saint Aubin, France,
dissolved in 0.9 NaCl solution. Each subject received intravenously a primed infusion of 0.7 mg kg
− 1
of tracer immediately followed by a 16 h constant infusion
of 0.7 mg kg
− 1
h
− 1
. Blood samples were drawn in tubes without anticoagulant but with a gel separator
Becton Dickinson, Meylan, France at 0, 0.25, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 14, 15 and 16 h after the primed
infusion. Serum was separated by centrifugation for 10 min at 4°C and 3000 × g. Sodium azide and aprotinin
were added to serum at final concentration of 500 and 17 mg l
− 1
, respectively.
2
.
3
. Analytical procedure
2
.
3
.
1
. Isolation of apolipoproteins VLDL density d B 1.006 g ml
− 1
and HDL 1.070 B d B 1.21 were isolated from plasma by se-
quential flotation ultracentrifugation, using a 50.4 rotor in a L7 apparatus Beckman Instruments, Palo Alto,
USA. HDL fractions were then dialyzed against a 10 mmol l
− 1
ammonium bicarbonate buffer pH 8.2 con- taining 0.01 EDTA and 0.013 sodium azide. VLDL
and HDL fractions were delipidated 1 h at − 20°C using ten volumes of diethylether-ethanol 3:1. Apolipo-
proteins were isolated by preparative discontinuous sodium dodecylsulfate SDS-polyacrylamide gel elec-
trophoresis on a 3 and 15 gel. The delipidated apoA-I and A-II containing material was solubilized in 0.05 M
Tris buffer pH 8.6, containing 3 SDS and 10 glyc- erol and applied to a 3-mm thick vertical slab gel. The
apoB-100 buffer contained 3 mercaptoethanol in ad- dition. After staining with Coomassie blue R-250,
apolipoproteins were cut from the gel and hydrolyzed in 6 N HCl for 16 h at 110°C under nitrogen vacuum.
Samples were then centrifuged to remove polyacry- lamide. Supernatants were lyophilized in a Speed Vac
Savant Instrument, Farmingdale, New York, USA. Lyophilized samples were dissolved in 50 acetic acid,
applied to an AG-50W-X8 200-400 mesh cation ex- change column Bio-Rad, Richmond, CA, and amino
acids were recovered by elution with 4 N NH
4
OH and lyophilized.
2
.
3
.
2
. Determination of leucine enrichment by gas chromatographycombustionisotope ratio mass
spectrometry GCCIRMS
Amino acids were converted to N-acetyl O-propyl esters and were analyzed on a Finnigan Mat Delta C
isotope ratio mass spectrometer Finnigan Mat, Bre- men, Germany coupled to a HP 5890 series II gas
chromatograph Hewlett
Packard. The
GC was
equipped with a splitsplitless injector and fitted with a BPX5 capillary column 30 m, 0.32 mm internal diame-
ter, 25 mm film thickness, S.G.E., Ringwood Vic., Aus- tralia and a 2 m retention gap RGK-1, SGE. Carrier
gas was helium and the column head pressure was set at 14 PSI. Injector temperature was 250°C for leucine
analysis. The splitless mode injection was adopted. The solvent purging valve was opened 0.6 min after injec-
tion. The column was held isothermal, at 50°C for 1 min after injection, then the temperature was pro-
grammed at 20°C min
− 1
up to 135°C, at 2°C min
− 1
from 135 to 149°C, at 15°C min
− 1
from 149 to 250°C, and was held for 5 min at 290°C. The operating condi-
tions of the ion source were as follows: source chamber pressure 1.4 × 10
− 6
mbar, ionising energy 80 eV, ion accelerating voltage 3 kV. Isotope abundance was ex-
pressed relatively to pulse peaks of reference gas. Data were analyzed using the supplier software Finnigan
ISODAT.
13
C leucine enrichment was initially expressed in delta ‰ and converted in tracertracee ratio prior modelling
[15 – 17].
2
.
4
. Modelling Kinetic data were analyzed with the simulation anal-
ysis and modelling SAAM II program SAAM Insti- tute, Inc., Seattle, WA. ApoA-I, A-II and B-100 data
were analyzed using the following monoexponential function: At = Ap1 − exp[ − kt − d], where At is
the apolipoprotein enrichment at time t, Ap the enrich- ment at the plateau of the VLDL apoB-100 curve, d the
delay between the beginning of the experiment and the appearance of tracer in apolipoproteins and k the frac-
tional synthetic rate of apolipoprotein [5,16,17]. It was assumed that the VLDL apoB-100 tracertracee ratio at
the plateau corresponds to the tracertracee ratio of the leucine precursor pool. It was also assumed that the
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
