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Sobenin et al. Is insulin atherogenic?

Reagents induce cholesterol accumulation in cultured cells),
Medium 199, fetal calf serum (FCS), penicillin, BALB/c mouse resident peritoneal macrophages were
streptomycin, Fungizone and L-glutamine were taken from non-stimulated animals as described by
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purchased from GIBCO Europe (Paisley, UK). Adams [22] . Afterward, 2 × 10 cells were seeded into
Collagenase type II was obtained from Worthington each well of 24-well plates in Medium 199 containing
Diagnostic System (Freehold, UK). [6- H]-thymidine antibiotics and 10% of FCS. After a 4-h incubation,
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(21 Ci/mmol) was purchased from Amersham the cultures were rinsed with Medium 199 to remove
International (Amersham, UK). Assay kits for total unattached cells. Then cells were incubated for
cholesterol determination were from Cell Biolabs, Inc. additional 4 h in Medium 199 containing 10% of
(San Diego, CA, USA). Other reagents were purchased examined patient’s serum. Lipid extraction, cellular
from Sigma-Aldrich Co., LLC (St. Louis, MO, USA). protein and cholesterol measurements were performed
as described above.
Cell culture
To study the direct effects of insulin on intracellular Statistical analysis
cholesterol accumulation and cellular proliferation, Results are reported as mean ± SEM, based on
cells were isolated from the non-atherosclerotic sub- triplicate measurements in each of 4 independent
endothelial (elastico-hyperplastic) sublayer of the experiments. Significance of differences was evaluated
intima by digestion of human aortic tissue with 0.15% using the IBM SPSS 20.0 statistical program package
collagenase under sterile conditions, as described and was assumed for P values < 0.05.
elsewhere [20] . The autopsy material was taken from
subjects aged 50 to 58 who had died suddenly by RESULTS
accident. After digestion, enzyme-isolated cells were
separated from the remaining tissue by filtering through Twenty-five, or 70% of 33 type 1 diabetic patient’s
nylon mesh, and subsequently resuspended in Medium sera exhibited an atherogenic potential, i.e. increased
199 containing standard additives: 2 mmol/L L-glutamine, total cholesterol content of cultured mouse peritoneal
100 U/mL penicillin, 100 µg/mL streptomycin, 2.5 µg/mL macrophages by 50-70% as compared to control. This
Fungizone and 10% of FCS. Cells were seeded into effect did not depend on patient’s age, gender, plasma
48-well or 24-well tissue culture plates at a density lipid levels, or diabetes duration. Moreover, serum-
of (3-4) × 10 cells/cm of growth area. The cells were induced cholesterol accumulation did not correlate
4
2
cultured at 37 ºC in a humidified CO -incubator (95% air with IRI level in tested serum samples (r = -0.09, P >
2
and 5% CO ). The primary cultures contained a mixed 0.6).
2
cell population made up primarily (95%) of typical and
modified smooth muscle cells as it was earlier defined In those 3 patients with newly diagnosed type 1 diabetes
by the ultrastructural features and immunofluorescent mellitus, blood serum obtained immediately before
markers. The medium was changed each day. On the the beginning of intensive insulin therapy induced
7th day in culture, Medium 199 containing 40% blood significant cholesterol accumulation in cultured mouse
serum from type 1 diabetic patients, 1 µCi/mL [ H]- peritoneal macrophages [Table 1]. As it was expected,
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thymidine and the indicated concentrations of insulin after the start of intensive insulin therapy, IRI level
(0.001-1,000 mU/mL), or Medium 199 containing increased rapidly, whereas C-peptide level remained
10% of FCS, 1 µCi/mL [ H]-thymidine and insulin low (the data not shown). By the end of the 1st day
3
(0.001-1,000 mU/mL) were applied to cell cultures. of intensive insulin therapy, blood glucose decreased to
After 24 h incubation, cells were rinsed 3 times with 10.5 ± 0.8 mmol/L, and IRI level accounted for 28
phosphate buffered saline (PBS), 3 times with PBS ± 3 mU/L. Further treatment resulted in the achievement
containing 0.2% bovine serum albumin, and again 3 of satisfactory glycemic control (data not shown). Along
times with PBS. Cellular lipids were extracted with the with these changes, the atherogenic effect of serum
mixture of n-hexane and isopropanol (3:2, vol:vol) as stayed practically invariable up to the 21st day of
described elsewhere [20] . Total cholesterol content was intensive insulin therapy, despite the steady decrease
determined enzymatically using commercial assay in blood glucose and elevation in IRI level [Table 1].
kits. After delipidation, the cells were rinsed 3 times Therefore, the ability of diabetic blood serum to induce
with 5% trichloroacetic acid and then were dissolved intracellular cholesterol accumulation was not directly
in 0.1 mol/L NaOH. Aliquots were used for cellular associated with either plasma insulin concentration or
protein determination and [ H]-thymidine incorporation the state of glycemic control.
3
as described by Tertov et al. [21] .
We have also studied the direct effect of insulin on
To measure serum atherogenicity (i.e. its ability to cho lesterol content and proliferative activity in cultured

176 Vessel Plus ¦ Volume 1 ¦ December 28, 2017