toneal macrophages [17] and in human macrophages isolated from atherosclerotic plaques [18] lipid loading
is accompanied by a marked decrease in expression and activity of lipoprotein lipase. Accelerated atherosclero-
sis is the main complication of diabetes. Also, diabetic state is known to induce metabolic activation in mono-
cytemacrophages [19] as well as enhanced phagocytic [20] and chemotactic [21] properties and increased cy-
tokine production [19]. To the best of our knowledge there are no reported data either on LPL expression
and secretion by monocyte-derived macrophages ob- tained from patients with non-insulin-dependent dia-
betes mellitus or about the loading of these cells with lipids. Therefore, in the present study, we tested a the
level of LPL mRNA expression and LPL activity secreted by monocytemacrophages from patients with
NIDDM versus non-diabetic subjects; and b the pos- sible correlation between LPL expression and activity
and the lipid loading of these macrophages in culture.
2. Materials and methods
2
.
1
. Patients The subjects chosen for the study fall into two cate-
gories, 1 non-insulin-dependent diabetics NIDDM, diagnosed for more than 1 year, with associated is-
chemic heart diseases stable angina pectoris and his- tory of myocardial infarction and; 2 patients
suffering from similar ischemia but without diabetes mellitus controls. A number of 30 NIDDM patients
and 24 non-diabetics controls ageing 40 – 70 years were selected from ‘D. Bagdasar’ emergency clinic in
Bucharest, with the personal patient consent. Patients were hospitalised for their coronary heart disease and
not for diabetes. However, diabetic patients were diag- nosed for at least 1 year and their diabetes was gener-
ally well controlled. For each patient the following parameters were assayed: serum cholesterol, HDL
cholesterol, triglycerides, glycemia, blood pressure and body mass index. Coronary atherosclerosis in both
groups of patients was documented by electrocardio- grams, exercise tolerance tests, and, in some cases, by
coronary angiography. The patients in the two groups were age and sex matched. Data are expressed as mean
values 9 S.D. and statistically analysed using the one- way ANOVA test for comparison between diabetic and
non-diabetic patients.
2
.
2
. Isolation and culture of monocyte-deri6ed macrophages
Mononuclear cells were isolated from whole blood, collected on 3.8 Na
2
citrate using Hystopaque 1077, according
to [22].
Monocytes were
subsequently purified by centrifugation using a Percoll gradient [23].
After two washes with Hank’s Balanced Salt Solution, the cells were seeded 10
6
cells per well in 24-well culture dishes in RPMI 1640 medium supplemented
with 20 autologous serum, 100 Uml penicillin and 100 mgml streptomycin. After 2 h incubation, the non-
adherent cells were washed and the remaining cells were cultured for 6 days, in 5 CO
2
humidified atmosphere, at 37°C, replacing the medium every 2 days. All the
assays were performed on the 7th day, after a 20 h incubation of cells with fresh medium. The cell viability
was evaluated using a LIVEDEAD EukoLight Viabil- ityCytotoxicity Kit Molecular Probes, Inc., Eugene,
OR. Briefly, after washing with warm medium, and fixation using 2 p-formaldehyde, cells were incubated
with both calcein stain for living cells and ethidium bromide homodimer stain for death cells, according to
the protocol indicated by the manufacturer. Finally, cells
were counted
under a
Nikon fluorescence
microscope.
2
.
3
. Lipoprotein lipase acti6ity assays One milliliter medium collected from four wells was
concentrated to a final volume of 100 ml using Cen- triPrep concentration tubes cut-off 10 000 Da, Mil-
lipore Intertech,
Marlborough, MA.
In some
experiments, the cells were previously treated with hep- arin 4 Ulml, for 20 min, in order to release the LPL
associated with the cell surface. The LPL activity was assayed by a non-radioactive method adapted from
Nilsson – Ehle and Schotz [24]. Briefly, 100 ml of concen- trated medium was incubated with 150 ml of a substrate
mixture, yielding a final concentration of 1.7 mM glyc- erol trioleate corrected according to each individual
plasma triglyceride values and 0.5 mM lecithin, 1 BSA in 80 mM Tris – HCl, NaCl 150 mM, pH 8.2 and
50 ml heat inactivated human serum, in 500 ml total volume. In all cases, the triglyceride hydrolysis does not
exceed 5 – 8 of the total amount of substrate in the mixture. Free fatty acids released in the reaction were
measured using the method of Itaya [25]. A control consisting from RPMI-1640 culture medium with 20
autologous serum was treated similarly to the corre- sponding probe and the activity value obtained was
subtracted from the latter. All measurements of LPL activity were performed within 2 h from medium
collection.
2
.
4
. Quantitati6e RT-PCR for LPL mRNA Total
RNA was
extracted from
10
4
to 10
6
macrophages after 7 days in culture. Cells were scraped from the plates using a rubber policeman, than washed
twice with serum-free medium and the total RNA was extracted using a GlassMax RNA Microisolation Spin
Cartridge System GIBCO BRL, Vienna, Austria ac- cording to the instructions provided by the manufac-
turer. The method is a modification of the procedure described in [26], suitable for a quantity as low as 10
4
cells. The total RNA obtained had a A
260
A
280
ratio between 1.8 and 2.0 and was immediately frozen and
kept at − 70°C until use. Quantitation of LPL mRNA was essentially performed according to [27]. Reverse
transcription was accomplished for 1 h at 37°C using 100 – 300 ng total cellular RNA, 10
6
copies of pAW109 cRNA Perkin-Elmer Corp., Norwalk, CT as an inter-
nal standard in a total volume of 20 ml, containing 0.25 m
g random hexamer primers, 0.5 mM dNTP, 50 U MMLV-RT-ase RT-PCR kit purchased from Strata-
gene Cloning Systems, LaJolla, CA. After reverse tran- scription, four serial 1:2 or 1:3 dilutions of the entire
cDNA mixture were amplified using a PCR Core Kit from Boehringer Mannheim GMBH Germany. The
oligonucleotide primers for LPL used in the reaction ordered
from MWG
Germany were,
5- GAGATTTCTCTGTATGGCACC-3 5primer and
5-CTGCAAATGAGACACTTTCTC-3 3
primer. Whenever the case, the 5 primers were labelled in the 5
end with [g-
32
P]ATP using polynucleotide kinase Boehringer Mannheim GMBH, Germany. The PCR
amplification protocol was carried out in a 100 ml volume, in 35 cycles involving denaturation at 95°, for
60 s, annealing at 60°, for 45 s and a final extension at 72°C, for 7 min, in a GeneAmp PCR System 2400
Perkin-Elmer Corp., Norwalk, CT. PCR products were separated by electrophoresis on 3 Nusieve 3:1
agarose gels FMS Bioproduct, Rockland, ME. Subse- quently to a 4 h migration, the PCR products were
stained directly in the agarose gel using the fluorescent stain SYBR Green II, as in [28]. Quantitative analysis
of the products was done by densitometry using a Shimadzu RF 5001 spectrometer and by plotting the
arbitrary units of fluorescence AUF against the inter- nal standard concentration [27]. Alternatively, some of
the probes were stained with ethidium bromide Sigma Chemical Co., St. Louis, MO, the appropriate bands
were excised from the gel and
32
P was quantitated by Cerenkov counting.
2
.
5
. Electron microscopy and morphometry To visualise the loading of macrophages with lipids
after 7 days in culture, the cells were washed three times with 100 mM phosphate buffer saline, pH 7.4 and fixed
in the culture plates with a mixture containing 3 para-formaldehyde, 5 glutaraldehyde, 2 osmium te-
traoxide in 1.1 M cacodylate buffer pH 7.4 and saturated solution of ferrous citrate on ice [29]. After 40
min, cells were scraped from the plates and pelleted by centrifugation at 5000 × g. After mordanting with 1
tannic acid for 10 min [29], the pelleted cells were dehydrated with increasing concentrations of ethanol
and embedded in Epon. Thin sections stained with uranyl acetate and lead citrate were examined with a
Phillips 400 Electron Microscope. Quantitative mea- surements of lipid droplet number were made directly
on electron micrographs of macrophages, in which the plane of the section was through the body of the cell. A
number of 20 cells for each experimental condition were randomly selected n = 5 per group.
2
.
6
. Lipid analysis To asses the intracellular lipid content, the cells
plated on four culture wells were washed three times with PBS and scraped with a rubber policeman in 1 ml
total volume of water. After a 30 s sonication of the cell suspension, lipids were extracted in a mixture of chloro-
form:methanol:water 2:2:1. The organic phase con- taining lipids was dried under nitrogen and the pellet
was solubilised in a chloroform:methanol 2:1 vv mix- ture. The amount of triglyceride and cholesterol free
and esterified was measured using enzymatic kits pro- vided by Sigma St. Louis, MO. Blanks and standard
curves for each lipid class were run in parallel. Intracel- lular lipids were expressed as mg lipid per mg cell
protein.
2
.
7
. Other assays Serum triglycerides, cholesterol and glycemia were
assayed by using Sigma enzymatic kits. HDL choles- terol was measured following precipitation with na-
trium phosphotungstateMgCl
2
as in
[30]. LDL
cholesterol was calculated according to Friedewald for- mula. Cell protein was determined according to Brad-
ford, as described in [31].
3. Experimental results
