International Journal of Clinical Medicine, 2011, 2, 231-245
doi:10.4236/ijcm.2011.23038 Published Online July 2011 (http://www.SciRP.org/journal/ijcm)
231
Modulation by Insulin of the Co-localized LDL
Receptor in Normal and Type-I Diabetic Subjects*
Shilpa Suneja1, Gopalakrishnan Ramakrishnan1, Nikhil Tandon2, Nimai Chand Chandra1#
1Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India;
2Department of Endocrinology & Metabolism, All India Institute of Medical Sciences, New Delhi, India.
Received March 22nd, 2011; revised May 17th, 2011; accepted June 17th, 2011.
ABSTRACT
Ongoing insulin therapy maintains LDL receptors at highly expressed state in Type-1 diabetic people; yet Type-1 dia-
betics are liable of having higher plasma LDL level. This disparity has raised doubt on the probability of existence of
functionally active LDL receptor in such people. Confocal microscopy and immunoprecipitation have made it evident
that a portion of insulin- and LDL receptors remain together in a co-localized mode, which only gets freed in presence
of insulin. The findings of this study have shown that insulin therapy protects Type-1 diabetic people from the patho-
genesis of atherosclerosis by decimating the inactivity of the co-localized LDL receptors in addition to its regular effect
of having increased glucose tolerance. The existence of co-localized state of these two receptors and their dependence
on insulin for independent activity has, at least, presented a reason for developing hypercholesterolemia and advanced
coronary atherosclerotic lesion in chronic Type-1 diabetic subjects.
Keywords: LDL Receptor, Insulin Receptor, Type-1 Diabetes, Atherosclerosis, Insulin, LDL
1. Introduction
Atherosclerosis, a consequence of poor LDL receptor
activity, is common in people with diabetes mellitus
(DM) [1]. Hyperlipoproteinemia, resulting from chronic
insulin-dependent diabetes mellitus (IDDM), may be
reversible provided it is effectively treated with insulin.
IDDM induced dyslipoproteinemia is not only a strong
risk factor for the development of atherosclerosis; it is
also one of the leading causes of specific microan-
giopathies [2,3]. Decreased LDL receptor sensitivity in
DM patients hampers the treatment and promotes pro-
gression of diabetic microangiopathies [4]. Patients of
type-2 DM (NIDDM), a defect of non-functionality of
insulin, are also prone to altered blood lipid and lipo-
protein profiles [5-14]. A study in Joslin clinic in Boston
between 1956 and 1968 [15] showed that about 78% of
diabetic patients die from Coronary Artery Disease
(CAD). Increased LDL level in blood is a well known
high risk factor for CAD. As LDL-cholesterol is a major
component of the atherosclerotic plaque, and since dia-
betics (both type-1 and type-2) are prone to developing
hypercholesterolemia; deficiency of insulin is expected
to play some role in generating hypercholesterolemia in
diabetic people. The increased transvascular LDL trans-
port in patients with type-1 DM suggests lipoprotein
influx into the arterial wall in people with type-1 DM,
possibly explaining accelerated development of athero-
sclerosis in people of type-1 DM [16].
It is known that insulin increases the LDL receptor
mRNA and receptor expression [17]. Although the exact
mechanism is not known, the increased LDL receptor
expression by added insulin, in an in vitro model ex-
periment, has been found to be regulated by the known
sterol regulated feedback mechanism in cells [18]. LDL
receptor is considered as one of the major cell surface
receptor protein responsible for plasma cholesterol
clearance and maintenance of intracellular cholesterol
homeostasis [19]. Although it is known that insulin
cannot stimulate LDL receptor expression in sterol satu-
rated cells [18], a consequence in atherosclerotic patho-
genesis; no direct evidence, so far available, of the role
of insulin in LDLR function in such cells. In diabetes
mellitus the stimulatory effect of insulin on LDL recep-
tor gene transcription is absent or meager [20]. However,
*S.S. was supported by a Scholarship Award from Indian Council o
f
Medical Research (ICMR), India; G.R. was supported by a Senio
r
Research Fellowship from the Council of Scientific and Industrial
Research (CSIR), India.

Modulation by Insulin of the Co-localized LDL Receptor in Normal and Type-I Diabetic Subjects
232
it is not clear whether this is the only reason for de-
creased LDLR function in diabetes and its improvement
with insulin administration.
Epidemiological studies show that in most of the
Type-I Diabetes Mellitus have increased atherosclerosis
and Type-1 diabetes is a state of pancreatic insufficiency
in insulin production. Since there is already a report on
the profile of LDL receptor expression in Type-2 pa-
tients of DM [21]; an attempt has been made in this
study to explore the possible mechanisms involved in
poor LDLR function in patients of Type-I DM. We have
studied the localization of two receptors, LDLR and IR
(insulin receptor), by confocal microscopy in monocyte
cells of normal human subjects and patients of Type-1
diabetes as well as in THP-1 cells. This shows that the
two receptors normally exist in co-localized state in an
un-stimulated situation. We have shown that insulin,
either secreted after a meal or administered in Type-I
diabetic subjects or even applied in the medium of
THP-1 cells, disrupts their co-localized association. Up-
take of LDL by monocytes is also increased in presence
of insulin. Our results have shown that the existence of
LDLR-IR co-localization and their dissociation by insu-
lin is a regulatory mechanism in monitoring the LDL
receptor function. The atherosclerotic complication in
Type-I diabetes thus may be a consequence of lack of
insulin to disrupt the co-localized state of LDLR-IR
complex.
2. Experimental Procedure
2.1. Materials and Reagents
Lymphoprep™ was purchased from Axis. Shield Poe AS,
Oslo, Norway. Antibodies against LDLR (goat poly-
clonal IgG) and IR (rabbit polyclonal) as well as fluores-
cent antibodies, fluorescein isothiocyanate (FITC)-la-
beled goat anti-rabbit IgG for Insulin receptor and phy-
coerythrin(PE)-labeled rabbit antigoat IgG for LDLR,
were purchased from Santa Cruz Biotechnology, Inc.
California, USA (DAKOLSAB + Kit). Kits for choles-
terol, HDL, LDL and Triglyceride estimation were ob-
tained from Giess Diagnostic’s snc, Via Crevinara,
Rome, Italy. Kit for Glycated haemoglobin estimation
was procured from from Life chem™ GHb, Kamineni
Life Sciences Pvt.Ltd., Hyderabad. Kit for Glucose es-
timation was obtained from DiaSys Diagnostic Systems
GmbH, Holzheim, Alemania. Ethylene diamine tetra
acetate (EDTA) as well as Antibiotic/Antifungal solu-
tion (100×) was purchased from Sigma Chemical Co., St.
Louis, MO, USA. RPMI-1640 powder was obtained
from GIBCO BRL, Life Technologies, Inc. Grand Island,
NY, USA. DAB Substrate Kit for Peroxidase and Strep-
tavidin Peroxidase Kit were bought from Vector, Labo-
ratories, Inc., Burlingame, CA, U.S.A. The Plasma for
LDL extraction was obtained from the blood bank at All
India Institute of Medical Sciences (AIIMS), New Delhi,
India. All other chemicals used were of analytical re-
agent grade.
2.2. Subjects
Only male subjects (>20 years, 15 control and 15 dia-
betic), control and treated Type-1 diabetic, were in-
cluded in the study following stipulated guidelines of the
Ethical Clearance Committee of AIIMS, New Delhi,
India.
2.3. Sample Collection
10 - 12 ml of blood samples were drawn aseptically
from the superficial veins of each of the study subjects.
Whole blood was used for monocyte isolation and esti-
mation of glycosylated hemoglobin. Plasma was used
for glucose estimation and serum was used for rest of the
studies.
Plasma and serum was separated from whole blood by
routine laboratory protocol.
2.4. Blood Monocyte Isolation
Blood monocytes were isolated according to the Com-
pany provided protocol (Sigma-Aldrich, Histopaque-
1077. Procedure No. 1077).
2.5. Preparation of LDL from Human Blood
Plasma
LDL was collected from human blood plasma (obtained
from the store of AIIMS Blood Bank hospital facility)
by NaCl-KBr density gradient ultracentrifugation ac-
cording to Havel et al. [22]. The LDL density band was
collected and dialysed against PBS (phosphate buffer
saline, pH 7.2) at 4˚C for 24 h and total cholesterol was
estimated as reported earlier [23].
2.6. LDL Uptake Study by Blood Monocytes
12-well plates were used for LDL uptake study. PBMC
were isolated from fasting blood. Cells were counted in
the Neubauer chamber and 2 × 105 cells were put in each
well along with 1ml of RPMI medium containing anti-
mycotic-antibiotic(1×) (Sigma, USA) but no serum.
Cells were incubated for one and half hour at 37˚C.
Wells were then washed with the serum free RPMI me-
dia with antimycotic-antibiotic supplements. The cells
were then incubated with different concentrations of
LDL in serum free medium for 5 h. After 5 h, the left-
over LDL concentration in the medium was measured to
find the amount of LDL taken up the cells.
Standard curve was made for LDL uptake by mono-
cytes from the control group’s blood samples using 0, 5,

Modulation by Insulin of the Co-localized LDL Receptor in Normal and Type-I Diabetic Subjects233
10, 15, 20, 30, 40, 60 and 80 µg cholesterol/ml culture
medium. .
Four concentrations―0, 20, 40 and 80 µg choles-
terol/ml were selected to compare the uptake pattern
between diabetic and control subjects.
2.7. Immunocytochemistry on Blood Monocytes
Isolated monocytes were grown on cover slips in 12-
well plates and used for immunocytochemistry as de-
scribed previously [23].
2.8. Confocal Microscopy
1) Human monocytes—Isolated monocytes from PBMC
were grown on cover slips kept under RPMI-1640
within the wells of a 12-well plate. The cells grown on
the cover slips were fixed in absolute acetone at 4˚C for
10 min. Cover slips were washed thrice with 0.01% Tri-
ton-X containing phosphate-buffer-saline (PBST) for
5min each. Blocking was then carried out in 1% BSA at
room temperature for 1 h. The cells were then washed
with PBST at room temperature. All cover slips were
then incubated with one antibody, either LDLR [goat
polyclonal (1:25)] or IR [rabbit polyclonal (1:25)] for 2
h at room temperature or overnight at 4˚C in a humid
chamber. PBST wash was given. The steps henceforth
were carried in dark. Fluorescent secondary antibody
(antirabbit goat IgG-FITC diluted 1:50 for insulin re-
ceptor or antigoat rabbit IgG-PE diluted 1:50 for LDLR)
was applied on cover slips and left for 1 hour incubation
at room temperature. PBST wash was given as before.
Incubation with second primary antibody on all the
cover slips was done next and all the following steps
described above were repeated again. The cover slips
were mounted in glycerol: PBS: 1:1 and then visualized
under confocal microscope. Image capturing was done
within next 24 h on a Leica confocal microscope at the
magnification of 400×.
2) THP-1 cells—THP-1 cells were seeded onto 12
mm cover glasses in a 6-well plate @ 5 × 105 cells/well
and grown 24 h in 50nM PMA (required to induce cell
adherence) containing RPMI-1640 medium supple-
mented with 10% fetal bovine serum and antibi-
otic-antimycotic mixture (1× final concentration) in
presence of 95% air and 5% CO2 at 37˚C. Following this
the medium was removed and the cells were serum
starved in the medium for 12 h. After serum starvation,
the medium was removed and the cells were washed
with ice-cold PBS and then fixed in absolute acetone at
4˚C and then again washed with PBS. Blocking was then
carried out in 1% BSA at room temperature for 1 hour.
Rest of the procedure was same as described with hu-
man monocytes above. The images were then captured
within next 24 h on a Leica confocal microscope at the
magnification of 400×.
For insulin treatment, the serum starved cells were
placed in 2 ml of ice-cold medium containing 15 µg/ml
insulin and incubated at 4˚C for 1 h. Following this, the
medium was replaced with fresh medium at 37˚C and
incubated for 10 min.
2.9. Estimation of Glucose, Total Cholesterol,
HDL, LDL, Triglyceride and Glycosylated
Hemoglobin
Respective Kits were used to estimate concentrations of
glucose, total cholesterol, HDL, LDL and triglyceride in
plasma/serum isolated from fasting male subjects. The
glycosylated hemoglobin was estimated in the whole
blood from same fasting male subjects.
Kit for Glucose estimation was from DiaSys Diagnos-
tic Systems GmbH, Holzheim, Germany.
Kits for Cholesterol, HDL, LDL and Triglyceride es-
timation were from Giess Diagnostics Inc, Via Crevinara,
Rome, Italy.
Kits for Glycosylated haemoglobin estimation was
from Life Chem™ GHb, Kamineni Life Sciences
Pvt.Ltd., Hyderabad, India.
2.10. Estimation of Insulin and C-Peptide
Insulin and C-peptide levels in serum were estimated
from the facility of the Department of Endocrinology
and Metabolism, AIIMS, New Delhi, India. In brief,
C-peptide was done by an immunoassay format and in-
sulin estimation was carried out following an immu-
nometric format on an ELECSYS 2010 auto-analyzer
(ROCHE) using an electrochemiluminiscence assay.
Minimum detectability for C-peptide was 0.01 ng/ml
and for insulin was 0.2 µU/ml.
2.11. THP-1 Cell Culture
Cells were grown to approximately 90% confluence in
RPMI-1640 medium supplemented with 10% fetal bo-
vine serum and antibiotic-antimycotic mixture (1× final
concentration) (Sigma, USA) in presence of 95% air and
5% CO2 in a 37˚C incubator. The medium was removed
and cells were grown in serum deficient medium for
another 12 h to stimulate receptor protein expression.
The cells were then used to prepare cell lysate by incu-
bating in cell lysis buffer, containing 50 mM Tris-HCl
(pH 7.6), 300 mM NaCl, 0.5% Triton-X-100, 5 mM
EDTA with 2 mM PMSF and 10 U/ml aprotinin added
just before use by vortexing strongly till the consistency
of the solution was changed. This lysed suspension was
kept on ice for 30 min and then spun at 10,000 g for 15
min at 4˚C. The supernatant was collected and protein
content was estimated.

Modulation by Insulin of the Co-localized LDL Receptor in Normal and Type-I Diabetic Subjects
234
2.12. Immunoprecipitation
THP-1 cell lysate containing 100 μg protein was mixed
with the anti-LDLR antibody (anti-human goat poly-
clonal, sc-11822 (N-17), Santa Cruz biotechnology, Inc.
USA) and incubated overnight at 4˚C. Protein A agarose
beads was blocked with 4% BSA for 2 h separately at
4˚C and then washed thrice with PBST. These blocked
beads were then added to the lysate antibody mixture
and kept at 4˚C for 2 h with constant mixing every 15 -
20 minutes. The beads were then collected by centrifu-
gation at 8000 × g for 5 minutes at 4˚C and washed with
chilled PBST. They were then re-suspended in protein
loading buffer and boiled for 5 minutes. The beads were
then pelleted by centrifugation and the supernatant was
used for SDS-PAGE.
2.13. Western Blotting/Immunoblot
The protein(s) from SDS-PAGE was transferred onto a
nitrocellulose membrane and developed with anti-hu-
man-insulin receptor β chain rabbit polyclonal antibody
(sc-711 (C-19), santa cruz biotechnology, Inc. USA)
diluted 1:8000 as reported previously [23].
2.14. Protein Estimation
Protein estimation was according to the method of
Bradford et al. using bovine serum albumin as the stan-
dard [24].
2.15. Statistics
Standard diviation was calculated and student’s t-test
was used to compare the means of two treatments. The
probability factor to judge the significance of the differ-
ence between the two means is shown as p value in the
parenthesis.
3. Results
3.1. Biochemical Parameters
Male subjects, normal and Type-1 diabetic, between 20
to 50 years of age were included in this study. Since
estrogen influences lipoprotein metabolism in females,
only male subjects were considered in this study. On an
average, the blood pressure, body weight and body mass
index (BMI) of the subjects (not shown) in our study
were maintained within limits to exclude the possibili-
ties of the mixed effects expected from other athero-
sclerotic inducers like hypertension, over-weight or obe-
sity. The biochemical parameters (Table 1) in this study
had made it evident that the subjects with a very high
plasma glucose (fasting concentration shown) and gly-
cated hemoglobin also had higher values in atherogenic
index [log(TG/HDL─C)], triglycerides (TG) and low
density lipoprotein (LDL) concentrations, which all are
Table 1. Biochemical parameters in Controls and Type-1
DM subjects. Atherogenic index [log(TG/HDL─C)] was
found considerably high in Type-1 diabetic subjects in ad-
dition to high plasma glucose and glycosylated Hb. LDL-
cholesterol and triglyceride concentrations were also found
noticeably high in diabetic patients as compared to the con-
trol group.
known risk factors to escalate atherosclerotic propensity
of an individual in course of time. Thus it was reflected
from the blood chemistry that subjects of Type-1 DM
had a high level of blood glucose accompanied by
dyslipidemia.
3.2. Receptor Expression
Immunocytochemistry was performed with monocytes
from fasting human plasma using respective antibodies
to evaluate the extent of expressions of insulin receptor
(IR) and LDL receptor (LDLR) in insulin treated Type-1
diabetic subjects (Figures 1(a) and (b)). Receptor ex-
pressions were judged in fifteen Type-1 diabetic subjects
against the expression profile of fifteen normal subjects.
Extent of receptor expression was estimated by integral
optical density (IOD) of the DAB stained receptors. The
graphical representation of the IOD of stained receptors
(Figure 1(c)) provided a direct comparison of the ex-
pression profile of IR and LDLR between Type-1 dia-
betic and control subjects. It was apparent from Figure
1(c) that both the receptor expressions (IR and LDLR)
were maintained at a higher level in insulin treated
Type-1 diabetic subjects as compared to the controls. It
was an interesting observation that in spite of having
high LDLR expression, the Type-1 diabetics were still
exhibiting a higher atherogenic index (Table 1).
PARAMETERS NORMAL
RANGE
NO.OF
SUBJECTS
CS DS
CONTROL
SUBJECTS
DIABETIC
SUBJECTS
1. PLASMA
GLUCOSE(F)
70 - 110
mg/dl
15 15 78.85
± 3.92
241.03
± 62.96
2. GLY. Hb. 3% - 5% 15 15 4.07
± 0.63
10.34
± 3.6
3. TOTAL CHO-
LESTEROL
<200
mg/dl
15 15 141
± 25.57
178.86
± 15.06
4. LDL-
CHOLESTEROL
66 - 178
mg/dl
15 15 93.84
± 20.23
114.28
± 34.06
5. HDL-
CHOLESTEROL
30 - 35
mg/dl
15 15 34.05
± 4.93
36.39
± 1.96
6. LDL/HDL 2 - 4 15 15 3.05
± 1.19
3.95
± 1.5
7.ATHEROGENIC
INDEX
[log(TG/HDL─C)]
<0.5 15 15 0.299
± 0.012
0.852
± 0.024
8.TGs
(Triglycerides)
36 - 115
mg/dl
15 15 83.47
± 23.40
119.31
± 17.68
9.LDL/TG 1.4 - 1.6 15 15 1.26
± 0.95
1.88
± 0.94

Modulation by Insulin of the Co-localized LDL Receptor in Normal and Type-I Diabetic Subjects235
(a)
(b)
(c)
Figure 1. The Figures (a) and (b) represent receptor expres-
sion of IR and LDLR in control and diabetic groups respec-
tively. The variations of IR & LDLR expression among
subjects of two groups (control/diabetic) have been evalu-
ated by estimating Integral Optical Density (IOD) of ex-
pressions (shown by bar graph in panel-c).
3.3. Functional Activity of LDLR
The LDL uptake profile of the expressed LDL receptors
on the surface of fasting human plasma monocytes, in
control and diabetic subjects, are shown in Figure 2.
The graphical representation in Figure 2(a) has
shown the LDL-cholesterol concentration taken up by a
population of monocytes isolated from 2 × 105 PBMC of
control fasting subjects. It increased almost linearly in a
rate controlled manner with the increased availability of
LDL-cholesterol till g/ml LDL cholesterol in the
culture medium (X-axis). Beyond g/ml LDL the
linearity discontinued but, uptake of LDL continued till
a concentration of g/ml LDL-cholesterol added in
the medium. When the monocytes were exposed to LDL
concentration beyond g/ml, they burst and showed
characteristics of foam cells (Figures 2(c)-(e) show in-
cubation with LDL up to 100 g/ml medium of the
monocytes isolated from control subjects).
In Figure 2(b) LDL-cholesterol uptake by plasma
monocytes has been compared between control and dia-
betic people. PBMC(s) were collected from fasting indi-
viduals only. Since sufficient blood samples were hardly
available from sick patients to study all nine concentra-
tions as tested in the samples from control subjects, only
four selected concentrations (0, 20, 40 and 80 µg/ml
medium) were chosen for this study to compare the up-
take rate of LDL-cholesterol between normal and dia-
betic subjects. Like control subjects, the uptake initially
increased in diabetic group in a linear fashion till a con-
centration of g/ml of LDL cholesterol in the me-
dium followed by a slower phase till a saturation of
g/ml LDL-cholesterol concentration in the culture
medium. However, at each point the uptake by diabetic
subjects was less than that of controls (p40 (S1,S2) < 0.05,
p80 (S1,S2) < 0.01). This low LDL receptor activity gave a
contrast impact to the highly expressed LDL receptors in
the diabetic subjects.
3.4. Co-localization Studies
Co-Immuno-Precipitation
LDL receptors were immunoprecipitated from the cell
lysate prepared from THP-1 cells incubated with and
without insulin. The immune-precipitate was probed
with anti-insulin-receptor-β-chain antibody after bloting
on nitro cellulose membrane (Figure 3(a)). The insulin
receptor band was absent in the insulin treated lane on
the nitrocellulose membrane (but present in the lane with
no insulin) after development with enhanced chemi-
luminisence (Santa Cruz Biotechnology, USA). This
showed that in absence of insulin the two receptors
co-immunoprecipitated but, insulin treatment separates
them apart and hence no band of insulin receptor (IR)
was found on nitrocellulose membrane because IR was
not co-immunoprecipitated with LDLR.
3.5. Confocal Microscopy
Confocal microscopy (Figure 3(b)) of the two receptors
(IR and LDLR) and their super imposition by computer
software made it apparent that the two receptors existed
in both free (red and green) and co-localized state (yel-
low) in monocytes isolated from fasting human blood of
normal subjects of varying age groups. The extent of
co-localization varied between individuals, irrespective
of age. More co-localization was expected to be a repre-

Modulation by Insulin of the Co-localized LDL Receptor in Normal and Type-I Diabetic Subjects
236
Figure 2. Panel-(a) (figure): The figure shows the uptake of LDL-cholesterol by monocytes in control subjects. Panel-(b) (fig-
ure): The figure shows the comparison of LDL-cholesterol uptake between normal subjects and diabetic patients. The figures
in Panel-(c), (d) and (e) show the comparison of monocytic cell (isolated from control subjects) lysis with increasing concen-
tration of LDL in the medium.
sentative of more inactive LDL receptors because
co-localized LDL receptors may exist in a less active
form. The processing protocol of cells for the confocal
microscopy had detergents (0.01% triton-X-100) to stop
nonspecific interactions on the cell surface by the com-
ponents of assay system. The permeabilization of plasma
membrane by detergent allowed antibodies to stain in-
tracellular receptors. Besides plasma membrane, the
evidence of colocalization was also seen in cytoplasm.
3.6. In-Vitro Model Study
Monocytes were isolated (see methods) from diabetic
subjects and the cultured monocyte cells were treated
with and without 15 µg of insulin/ml (concentration
found suitable for moderate LDL uptake in similar ex-
periments not shown here) culture medium for 10 min-
utes. The control cells (from diabetic subject and insulin
untreated) and insulin treated cells were processed for
confocal microscopy to see the effect of insulin on re-
ceptor colocalization. This experiment also showed the
separation of colocalized receptors by insulin (Figure
3(c)). When the experiment was repeated with THP-1
monocyte cells cultured in the laboratory, the same re-
sult was replicated (Figure 3(d)).
3.7. Non-Hyperglycemic Control Subjects
The extent of co-localization of IR and LDLR was stud-
ied in normal subjects (Figure 4), having no symptom of
hyperglycemia and without any family history of diabe-
tes, before and after of oral glucose administration. It
was expected that oral glucose would induce insulin
secretion resulting in reduction of co-localization of the
receptors. The plasma level of glucose, glycated-Hb
(Hb-A1c) and cholesterol were within normal limits in

Modulation by Insulin of the Co-localized LDL Receptor in Normal and Type-I Diabetic Subjects237
(a)
(b)
(c)

Modulation by Insulin of the Co-localized LDL Receptor in Normal and Type-I Diabetic Subjects
238
(d)
Figure 3. (a): LDL receptor was immunoprecipitated by anti-LDLR antibody from THP-1 cells treated with and without
insulin (15 µg/ml). After running on PAGE, the precipitate(s) was transferred on nitrocellulose membrane and treated with
anti-insulin receptor antibody. After development by enhanced chemi-luminisence (Santa Cruz Biotechnology, USA), an in-
sulin receptor band was found only in control panel.
all three volunteers participated in this study (Figure
4(d)). All three subjects showed normal GTT (glucose
tolerance test) (Figures 4 (a)-(c)). At 0.5 h, there was an
increased concentration of insulin in response to the glu-
cose consumed by these subjects after donating their fast-
ing blood. The changes of glucose and insulin concentra-
tion after each 0.5 h were the characteristics of the indi-
vidual’s own metabolic activity. The C-peptide level re-
mained constant from 0.5 h onwards indicating that no
further insulin was secreted after 0.5hr. The co-localized
state of the two receptors (LDLR and IR) in each subject
was found inversely co-related with the plasma insulin
level following glucose ingestion. A maximum separation
of the two receptors from their co-localized state (Figures
4 (a)-(c), confocal pictures) was found at 0.5 h when in-
sulin concentration was at its maximal height. The next
2hs follow up [Fasting and post glucose diet] showed that
the co-localized state of the two receptors increased as the
plasma insulin levels reduced. At 2 h, the co-localized
state of the receptors was close to the fasting pattern. Be-
cause of experimental compliance, the last 2hr data point
of the Subject-2 [C-2] is not available. Hence, this
co-relation study supports the role of insulin in generating
free LDLR from the IR-LDLR co-localized complex.
3.8. Diabetic Subjects
The co-localization of IR and LDLR was also studied in
three diabetic subjects after administering insulin. The
plasma level of glucose and glycated-Hb (Hb-A1c) were
noticeably high in all three subjects (Figure 5(d)). Sub-
jects DM-1 and DM-3 could maintain the normal limits
of total and LDL cholesterol. Subject DM-2 had a
markedly high value for both. Since a complete GTT
was unsuitable for these diabetic subjects, only three
time point were studied. The samples were taken: I) at
fasting state, II) half an hour after a meal taken and half
an hour after of insulin injection and III) immediately
before next insulin injection (about 4 h after the second
bleed). Since Type-1 diabetic people were deficient in
their in vivo insulin, the stability of the colocalized re-
ceptors was judged against the persistance of externally
added insulin in the blood plasma. The profile of all the
three parameters viz glucose, insulin and C-peptide in
blood plasma have been compared with the confocal
representation of IR-LDLR colocalized complex for
each diabetic subject (Figures 5(a)-(c), DM-1, DM-2
and DM-3). The Subject DM-1 showed that the
co-localized receptors in the fasting monocytes got
separated and became free after administration of exter-
nal Insulin which led to a fall in plasma glucose. The
initial extent of co-localization of DM-2 and DM-3 was
less than that of DM-1, probably because of the admini-
stration of intermediate long acting insulin at night.
However, even in these subjects the administration of
soluble insulin resulted in a marked decrease in
co-localization of the receptors as reflected in the second
sample (i.e. one hr after insulin injection and meal).
Co-localization was restored in the third sample taken 4
h later. This again showed that Insulin was responsible
for generating free receptors from the co-localized
IR-LDLR complex.
In Subject DM-3 the rate of fall was relatively slow
for insulin; the IR and LDLR also existed even longer in
free non-colocalized fashion.
4. Discussion
Type-1 diabetic patients are at increased risk of athero-
sclerosis and its clinical sequel. Retention of lipopro-
teins [25,26] in the arterial wall initiates the early stage
of atherosclerosis. This is then followed by activation of
endothelial expression of adhesion molecules [27-29],
development of cholesterol-laden foam cells [30,31] and
formation of atherosclerotic plaque [32-34]. The gly-

Modulation by Insulin of the Co-localized LDL Receptor in Normal and Type-I Diabetic Subjects239
(a)
(b)

Modulation by Insulin of the Co-localized LDL Receptor in Normal and Type-I Diabetic Subjects
240
(c)
(d)
Figure 4. The colocalization pattern of two receptors, LDL receptor and insulin receptor, were compared in three control
subjects with their blood glucose, insulin and C-peptide levels in fasting and prospandial state. The biochemical parameters
assayed in the fasting blood of those three controls are depicted in associated table.
cated lipoprotein(s) affects LDL receptor activity. Epi-
demiological data has firmly established the correlation
between diabetes and atherosclerosis. The present study
intends to find the possible reason(s) for developing
atherosclerosis in the people of Type-1 diabetes by
studying the inter-relation, if any, between insulin, insu-
lin receptor (IR)and LDL receptor (LDLR) in a model
system with peripheral blood mononuclear cells (PBMC)
isolated from Type-1 diabetic subjects and their age
matched controls.
Keeping in mind that LDL receptor function is com-
promised in DM, the present study tried to elucidate the
inter relationship between insulin activity and the ob-
served co-localization of IR and LDLR in normal and
diabetic subjects. The THP-1 monocyte cell line was
also used to verify the effect of insulin on the aggrega-
tion of two receptors in (IR and LDLR) in vitro studies.
Type-I diabetes was studied because the effects of abso-
lute insulin deficiency and its replenishment was easier
to determine. Only male subjects were included, so as to
rule out the confounding effects of estrogens. The rou-
tine therapeutic protocol of the Type-1 diabetic subjects
was not interrupted by any occasion of the present study.
In fact, the two groups (control and treated diabetic)
were found comparable except in terms of glycosylated
hemoglobin and lipid profiles (Table 1). We observed

Modulation by Insulin of the Co-localized LDL Receptor in Normal and Type-I Diabetic Subjects241
(a)
(b)

Modulation by Insulin of the Co-localized LDL Receptor in Normal and Type-I Diabetic Subjects
242
(c)
(d)
Figure 5. The colocalization pattern of two receptors, LDL receptor and insulin receptor, were compared in three diabetic
subjects under therapy with their blood glucose, insulin and C-peptide levels in fasting, one hour after insulin injection and
immediately before next insulin injection. The biochemical parameters assayed in the fasting blood of those three diabetic
subjects are depicted in associated table.
that the diabetic subjects, who were receiving their rou-
tine therapy, had higher IR and LDLR expression (Fig-
ure 1). But, LDL uptake was significantly lower in the
diabetic group (Figure 2). Hence co-localization of IR
and LDLR was studied even in more detail from the
perspective of insulin activity, in order to suggest a basis
for lowered LDLR activity in the diabetic subjects.
Since the LDL uptake study was performed on the
monocytes isolated from fasting blood samples before
any insulin application, the insulin treatment was not
expected to have any major influence on the assay sys-
tem. As Type-I diabetic subjects are in a persistently
insulin deficient state, increased co-localization in ab-
sent of insulin was taken as indicative of lowered LDLR
activity because of the lack of freely available inde-
pendent LDLRs.

Modulation by Insulin of the Co-localized LDL Receptor in Normal and Type-I Diabetic Subjects243
Co-localization of IR and LDLR was substantiated in
THP-1 cells, where IR-LDLR aggregation and its disag-
gregation in the presence of insulin has also been con-
firmed by demonstrating co-immunoprecipitation of
both receptors (Figure 3(a)). The co-immunoprecipita-
tion was abrogated in presence of insulin.
Confocal microscopy has demonstrated IR-LDLR
co-localization (Figure 3(b)), reduced by insulin in cul-
tured PBMCs and THP-1 cells (Figures 3 (c) and (d)).
In normoglycemic control subjects, induction of insulin
by oral glucose had the same effect; where maximum
disaggregation was noted with the peak of insulin level
in blood after oral glucose administration (Figure 4). In
the diabetic subjects the disaggregation was dependent
on externally administered insulin (Figure 5). The ab-
sence of any increase in C-peptide level in diabetic sub-
jects was an indicative of the lack of secretion of in vivo
biological insulin.
This study suggests that the IR-LDLR co-aggregation
in the absence of insulin could be a basis for the reduced
LDLR activity in diabetes. It is very well known that
two interacting proteins can exist in differential func-
tional states [35,36]. Here IR-LDLR is activated by dis-
sociation resulting from interaction of one of the part-
ners with its ligand(s). Although both the receptors can
bind their respective ligands simultaneously, other stud-
ies in our laboratory (not shown here) have shown the
priority of insulin at its level of 15 µg/ml culture me-
dium over LDL in dissociating the two receptors in short
interval.
To conclude, we are reporting, to best of our knowl-
edge, for the first time that a large proportion of LDLR
and IR interact with each other and are co-localized with
each other. This interaction is disrupted by insulin action.
There is a suggestion that this interacting LDLR-IR
complex has non/less-functional LDLR and could be
one of the possible mechanisms for poor LDLR func-
tioning in diabetes.
5. Acknowledgments
The confocal microscope facility in the department of
physiology, AIIMS, New Delhi, India is gratefully ac-
knowledged. We are greatly thankful to Professor J.
Sengupta (for giving access to the confocal microscope
facility in the department of physiology) and to Dr. A.K.
Dinda (for measuring I.O.D. of immunocytochemistry in
his facility in the department of pathology). We greatly
appreciate the help from Dr Nandita Gupta, department
of endocrinology and metabolism, for the estimation of
insulin and c-peptide in our patient’s samples. These
studies were supported by funding from the Indian
Council of Medical Research (ICMR), Government of
India; by the Department of Science and Technology
(DST), Government of India; by the Council of Scien-
tific and Industrial Research (CSIR), Government of
India and by the Institute Research Grant Support of All
India Institute of Medical Sciences (AIIMS), New Delhi,
India. The authors thank Prof Subrata Sinha, Head of the
department of biochemistry, for editing the manuscript
with necessary changes where needed.
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