Possible role of cytosolic free calcium concentrations in mediating insulin resistance of obesity and hyperinsulinemia

Abstract

Insulin- and glyburide-stimulated changes in cytosolic free calcium concentrations [( Ca2+]i) were studied in gluteal adipocytes obtained from six obese women (139 +/- 3% ideal body wt) and six healthy, normal weight age- and sex-matched controls. Biopsies were performed after an overnight fast and twice (at 3 and 6 h) during an insulin infusion (40 mU/m2 per min) (euglycemic clamp). In adipocytes obtained from normal subjects before insulin infusion, insulin (10 ng/ml) increased [Ca2+]i from 146 +/- 26 nM to 391 +/- 66 nM. Similar increases were evoked by 2 microM glyburide (329 +/- 41 nM). After 3 h of insulin infusion, basal [Ca2+]i rose to 234 +/- 21 nM, but the responses to insulin and glyburide were completely abolished. In vitro insulin-stimulated 2-deoxyglucose uptake was reduced by insulin and glucose infusion (25% stimulation before infusion, 5.4% at 3 h, and 0.85% at 6 h of infusion). In obese patients, basal adipocyte [Ca2+]i was increased (203 +/- 14 nM, P less than 0.05 vs. normals). The [Ca2+]i response demonstrated resistance to insulin (230 +/- 23 nM) and glyburide (249 +/- 19 nM) stimulation. Continuous insulin infusion increased basal [Ca2+]i (244 +/- 24 nM) and there was no response to either insulin or glyburide at 3 and 6 h of study. Rat adipocytes were preincubated with 1-10 mM glucose and 10 ng/ml insulin for 24 h. Measurements of 2-deoxyglucose uptake demonstrated insulin resistance in these cells. Under these experimental conditions, increased levels of [Ca2+]i that were no longer responsive to insulin were demonstrated. Verapamil in the preincubation medium prevented the development of insulin resistance.

Full text

Possible
Role
of
Cytosolic
Free
Calcium
Concentrations
in
Mediating
Insulin
Resistance
of
Obesity
and
Hyperinsulinemia
B.
Draznin,
K.
E.
Sussman,
R.
H.
Eckel,
M.
Kao,
T.
Yost,
and
N.
A.
Sherman
The
Research
Service
and
Department
of
Medicine,
Veterans
Administration
Medical
Center;
and
The
University
of
Colorado
Health
Sciences
Center,
Denver,
Colorado
80220
Abstract
Insulin-
and
glyburide-stimulated
changes
in
cytosolic
free
cal-
cium
concentrations
(QCa2+Ii)
were
studied
in
gluteal
adipocytes
obtained
from
six
obese
women
(139±3%
ideal
body
wt)
and
six
healthy,
normal
weight
age-
and
sex-matched
controls.
Biopsies
were
performed
after
an
overnight
fast
and
twice
(at
3
and
6
h)
during
an
insulin
infusion
(40
mU/m2
per
min)
(eu-
glycemic
clamp).
In
adipocytes
obtained
from
normal
subjects
before
insulin
infusion,
insulin
(10
ng/ml)
increased
[Ca2+ji
from
146±26
nM
to
391±66
nM.
Similar
increases
were
evoked
by
2
tiM
glyburide
(329±41
nM).
After
3
h
of
insulin
infusion,
basal
ICa2+ii
rose
to
234±21
nM,
but
the
responses
to
insulin
and
glyburide
were
completely
abolished.
In
vitro
insu-
lin-stimulated
2-deoxyglucose
uptake
was
reduced
by
insulin
and
glucose
infusion
(25%
stimulation
before
infusion,
5.4%
at
3
h,
and
0.85%
at
6
h
of
infusion).
In
obese
patients,
basal
adipocyte
[Ca2+Ii
was
increased
(203±14
nM,
P
<
0.05
vs.
normals).
The
[Ca2+ji
response
demonstrated
resistance
to
insulin
(230±23
nM)
and
glyburide
(249±19
nM)
stimulation.
Continuous
insulin
infusion
in-
creased
basal
[Ca2+1i
(244±24
nM)
and
there
was
no
response
to
either
insulin
or
glyburide
at
3
and
6
h
of
study.
Rat
adipocytes
were
preincubated
with
1-10
mM
glucose
and
10
ng/ml
insulin
for
24
h.
Measurements
of
2-deoxyglu-
cose
uptake
demonstrated
insulin
resistance
in
these
cells.
Under
these
experimental
conditions,
increased
levels
of
[Ca2+Ii
that
were
no
longer
responsive
to
insulin
were
demon-
strated.
Verapamil
in
the
preincubation
medium
prevented
the
development
of
insulin
resistance.
Introduction
The
role
of
intracellular
calcium
as
a
mediator
of
insulin
ac-
tion
was
originally
proposed
by
Clausen
et
al.
in
1974
(1)
and
by
Kissebah
et
al.
in
1975
(2).
Since
then,
considerable
evi-
dence
favoring
this
hypothesis
has
been
accumulated
(3-5).
Although
some
investigators
failed
to
observe
a
relationship
between
calcium
and
insulin
action
(6,
7),
diverse
aspects
of
insulin
action
have
been
demonstrated
to
be
dependent
upon
extracellular
and
cytoplasmic
Ca2+
(8-14).
Address
reprint
requests
to
Dr.
Boris
Draznin,
Veterans
Administra-
tion
Medical
Center,
Box
111-H,
1055
Clermont
Street,
Denver,
CO
80220.
Receivedfor
publication
12
April
1988
and
in
revisedform
I
July
1988.
Using
a
new
calcium
indicator
(fura-2),
we
recently
dem-
onstrated
that
insulin
and
glyburide
are
capable
of
increasing
cytosolic
free
calcium
concentrations
((Ca2+]i)'
in
isolated
rat
adipocytes,
primarily
by
enhancing
Ca2+
transport
across
plasma
membranes
(15).
In
the
present
study
we
have
attempted
to
answer
three
questions:
(a)
do
insulin
and
glyburide
increase
[Ca2+]i
in
adi-
pocytes
obtained
from
normal
subjects;
(b)
are
similar
effects
of
insulin
and
glyburide
observed
in
adipocytes
isolated
from
patients
with
moderate
obesity;
and
(c)
does
in
vivo
hyperin-
sulinemia
alter
the
cellular
response
to
insulin
and
glyburide
stimulation.
In
particular,
we
focused
on
the
role
of
[Ca2+]i
in
modulating
cellular
sensitivity.
The
latter
studies
were
per-
formed
during
a
6-h
insulin
infusion
(euglycemic
clamp).
Methods
Materials.
Porcine
insulin
was
a
gift
from
Eli
Lilly
Co.
(Indianapolis,
IN)
and
glyburide
was
generously
supplied
by
Upjohn
Co.
(Kalama-
zoo,
MI).
Fura-2
and
fura-2AM
were
purchased
from
Behring
Diag-
nostics
(San
Diego,
CA)
and
collagenase
was
obtained
from
Worth-
ington
Biochemical
Corp.
(Freehold,
NJ).
Experimental
design.
Gluteal
adipose
tissue
was
obtained
by
needle
biopsy
in
six
obese
women
(139±3%
ideal
body
wt)
and
six
healthy,
normal
weight
age-
and
sex-matched
controls.
The
biopsies
were
per-
formed
after
an
overnight
fast
and
twice
(at
3
and
6
h)
during
insulin
infusion
(40
mU/M2
per
min)
(euglycemic
clamp)
as
previously
de-
scribed
(16).
Blood
glucose
levels
in
all
patients
and
control
subjects
were
maintained
in
the
range
of
85-95
mg/dl.
Adipocytes
were
isolated
by
the
method
of
Rodbell
(17).
Obese
subjects
were
studied
before
and
3
mo
after
moderate
weight
loss
(down
to
127±4%
ideal
body
wt).
During
these
3
mo
they
were
maintained
on
an
isocaloric
weight
main-
tenance
diet.
Because
weight
reduction
did
not
alter
basal
or
insulin-
and
glyburide-stimulated
[Ca2+]i,
the
results
were
combined
for
the
sake
of
clarity.
Measurements
of
[Cai']i.
These
measurements
were
performed
as
previously
described
(15,
18)
using
a
spectrofluorometer
(model
340;
Turner
Designs,
Mountain
View,
CA).
During
fura-2
loading
(45
min
at
37°C)
and
Ca2+
measurements
the
cells
were
incubated
in
2.4
ml
of
Krebs-Hepes
buffer
containing
1
mM
CaCl2,
118.4
mM
NaCI,
4.69
mM
KCI,
1.2
mM
MgC12,
1.18
mM
KH2PO4,
1.25
mM
NaHCO3,
20
mM
Hepes,
5
mg/ml
BSA,
and
30
mg/dI
glucose
at
pH
7.4.
The
final
cell
concentration
was
-
2
X
I0O
cells/cuvette
(or
8
X
I
04cells/ml)
and
the
measurements
were
obtained
before
and
10
min
after
additions
of
either
insulin
(10
ng/ml)
or
glyburide
(2
,M).
The
fluorescence
of
the
extracellular
fura-2
was
estimated
by
adding
MnCI2
(50
MM),
which
quenches
extracellular
fura-2.
MnCI2
was
then
chelated
by
the
addition
of
100
MM
pentetic
acid.
The
estimate
of
extracellular
fura-2
was
made
before
stimulation
of
the
cells
with
insulin,
glucose,
or
glyburide.
The
fluorescence
of
either
the
buffers
used
in
these
studies
or
of
tissues
(without
fura-2)
was
10-13%
of
that
observed
with
the
cells
loaded
The
Journal
of
Clinical
Investigation,
Inc.
Volume
82,
December
1988,
1848-1852
1.
Abbreviations
used
in
this
paper:
[Ca2+]i,
cytosolic
free
calcium
concentrations.
1848
Draznin,
Sussman,
Eckel,
Kao,
Yost,
and
Sherman

C
4001-
T
3001
C
c)
200[
100
01
Insulin
(10
ng/ml)
Glyburide
(2
pM)
_I+
_
+
+
_
_
~+
with
the
probe.
Cellular
and
buffer
fluorescence
did
not
change
in
response
to
either
glucose,
insulin,
or
glyburide.
Results
are
presented
as
mean±SEM
and
compared
using
paired
or
unpaired
t
tests.
2-Deoxyglucose
uptake.
Adipocytes
(2
X
l0
cells)
were
incubated
in
the
absence
and
in
the
presence
of
insulin
(25
ng/ml)
for
30
min
at
370C.
Glucose
uptake
was
initiated
by
the
addition
of
[3H]2-deoxyglu-
cose
(0.2
PCi).
After
3
min
of
incubation
the
reaction
was
terminated
by
transferring
200-Al
aliquots
of
incubation
mixture
to
the
microfuge
tubes
containing
100
gl
silicone
oil
and
centrifuging
the
tubes
in
a
microfuge
(Beckman
Instruments,
Inc.,
Palo
Alto,
CA).
The
cell
pellets
were
counted
for
radioactivity
present
in
a
liquid
scintillation
counter
(Beckman
Instruments,
Inc.).
In
vitro
studies
with
rat
adipocytes.
Adipocytes
isolated
by
the
method
of
Rodbell
(17)
were
preincubated
for
24
h
as
previously
described
by
Marshall
et
al.
(19)
and
Garvey
et
al.
(20).
The
incubation
media
contained
either
1
or
10
mM
glucose
with
or
without
10
ng/ml
insulin.
After
preincubation,
the
cells
were
washed
three
times
and
incubated
for
40
min
in
glucose-
and
insulin-free
medium
to
eliminate
any
possible
influence
of
high
glucose
or
insulin
present
during
the
24-h
preincubation.
The
cells
were
resuspended
in
KRB
and
divided
into
two
groups
for
2-deoxyglucose
uptake
and
[Ca2"]i
determinations
as
described
above.
Results
The
basal,
nonstimulated
level
of
[Ca2"]i
in
adipocytes
ob-
tained
from
the
control
subjects
before
insulin
infusion
was
146±26
nM.
Incubation
of
these
adipocytes
with
insulin
(10
ng/ml)
for
10
min
at
37°C
resulted
in
an
increase
in
[Ca2+]i
to
391±66
nM
(Fig.
1
A).
A
similar
increase
was
evoked
by
2
,uM
glyburide
(329±41
nM).
In
adipocytes
obtained
after
3
h
of
insulin
infusion
(40
mU/M2
per
min),
basal
[Ca2+]i
rose
to
234±21
nM
(Fig.
1
B),
but
responses
of
these
adipocytes
to
both
insulin
and
glyburide
were
completely
abolished
(I197±13
nM
and
224±5
nM,
respectively).
[Ca2+]i
continued
to
in-
crease
during
insulin
infusion,
reaching
255±24
nM
at
the
6th
h
of
the
infusion
(Fig.
1
C).
There
was
no
further
stimulation
by
either
insulin
or
glyburide
(214±22
nM
and
235±40
nM,
respectively).
To
examine
how
this
acquired
inability
of
insulin
to
in-
crease
[Ca2+]i
related
to
insulin
action
on
glucose uptake,
we
studied
basal
and
insulin-stimulated
2-deoxyglucose
uptake
in
the
fat
cells
obtained
from
three
normal
individuals
at
0,
3,
and
6
h
of
euglycemic
clamp.
The
gluteal
biopsies
were
ob-
tained
in
the
manner
identical
to
those
used
to
study
[Ca2+]i.
Figure
1.
Effect
of
insulin
and
glyburide
on
[Ca2"]i
in
adipocytes
obtained
from
six
normal
women
after
overnight
fast
(A),
at
the
3rd
(B)
and
6th
(C)
h
of
euglycemic
clamp.
Re-
sults
represent
mean±SEM.
Control
values
for
[Ca2+]i
repre-
sent
time
controls;
that
is,
the
levels
of
[Ca2"]i
at
0,
3,
and
6
h
of
clamp
but
before
in
vitro
additions
+
-
of
either
insulin
or
gly-
-
-
+
buride.
Before
insulin
and
glucose
infusion,
insulin
increased
in
vitro
2-deoxyglucose
uptake
by
24%
(Fig.
2).
At
the
3rd
and
6th
h
of
euglycemic
glucose
clamp,
the
unstimulated
(basal)
levels
of
2-deoxyglucose
uptake
were
unchanged,
but
insulin-stimu-
lated
values
were
significantly
reduced
when
compared
with
preclamp
studies
(Fig.
2).
In
obese
patients
before
insulin
infusion,
basal
adipocyte
[Ca2+]i
increased
compared
with
normal
subjects
(203±14
nM,
P
<
0.05).
The
[Ca2+]i
response
of
these
adipocytes
dem-
onstrated
resistance
to
both
insulin
(230±23
nM,
12%
in-
crease)
and
glyburide
(249±19
nM,
16%
increase)
(Fig.
3).
The
comparison
of
these
results
with
those
obtained
in
normal
individuals
is
shown
in
Fig.
4.
Continuous
infusion
of
insulin
in
obese
patients
further
increased
basal
[Ca2+]i
(244±24
nM
at
3
h
and
252±32
nM
at
6
h)
and,
similar
to
the
controls,
there
was
no
response
of
the
isolated
adipocytes
to
either
insulin
or
glyburide
at
3
and
6
h
of
study.
If
glucose
and/or
insulin
infusion
induce
insulin
resistance
in
adipocytes
within
3
h,
this
phenomenon
should
be
repro-
ducible
in
vitro.
To
examine
this
possibility,
we
incubated
freshly
isolated
rat
adipocytes
for
24
h
in
the
presence
of
either
1
or
10
mM
glucose
with
or
without
10
ng/ml
insulin.
A
similar
experimental
approach
was
previously
used
by
Garvey
et
al.
(20),
who
studied
rat
adipocytes,
and
by
Sinha
et
al.
(21),
who
studied
human
adipocytes.
After
a
24-h
preincubation
period,
the
adipocytes
were
washed
with
insulin-
and
glucose-
free
medium
to
deactivate
the
glucose
transport
system.
These
cells
were
then
used
to
determine
2-deoxyglucose
uptake
and
0
0
o
0
0
10
0'
_
x
11
O
4
0
0
C4
0
O
co
E
L
Before
Euglycomic
Clamp
Figure
2.
Insulin-stimu-
lated
2-deoxyglucose
uptake
in
adipocytes
obtained
by
gluteal
biopsy
from
three
nor-
mal
individuals
before
and
during
euglycemic
clamp.
Results
repre-
3
h
6
h
sent
mean±SEM
of
of
Clump
of
Clamp
three
determinations.
Cytosolic
Free
Calcium
Concentrations
and
Insulin
Resistance
1849
A

M
r-T
150
100
50
-
Insulin
(10
ng/ml)
Glyburide
(2
pM)
y0
o
c
O
In
-Jo
aD
E
0
W
a'
Co
+_
_-
+
0.90
0.80
0.70
0.60
0.50
0.40
0.30
0.20
0.10
0.00
24-HOUR
PREINCUBATION
1
mM
Glucose
10
mU
Glucose
-
basal
I
E221insulin
25ng/ml
0
10
0
10
PREINCUBATION
INSUUN
CONCENTRATION
(ng/ml)
Figure
5.
Effect
of
24-h
preincubation
with
1
and
10
mM
glucose
(with
or
without
10
ng/ml
insulin)
on
basal
and
insulin-stimulated
2-
deoxyglucose
uptake
in
rat
adipocytes.
Results
represent
the
mean±SEM
of
five
to
eight
experiments.
A%,
%
stimulation
by
insulin.
Figure
3.
Effect
of
insulin
and
glyburide
on
[Ca2"]i
in
adipocytes
ob-
tained
from
six
obese
women
after
overnight
fast.
Results
represent
mean±SEM.
[Ca2+]i.
In
adipocytes
preincubated
with
1
mM
glucose,
both
in
the
presence
and
in
the
absence
of
insulin,
subsequent
ad-
dition
of
insulin
stimulated
2-deoxyglucose
uptake
by
-
100-115%
(Fig.
5).
The
presence
of
insulin
(10
ng/ml)
in
the
24-h
preincubation
media
did
not
alter
either
basal
or
insulin-stimulated
2-deoxyglucose
uptake.
In
contrast
to
preincubations
with
1
mM
glucose,
the
pres-
ence
of
10
mM
glucose
for
24
h
significantly
reduced
insulin-
stimulated
2-deoxyglucose
uptake
(P
<
0.01).
Insulin
in
the
preincubation
media
reduced
both
basal
and
insulin-stimu-
lated
glucose
uptake
(Fig.
5).
To
examine
whether
this
glucose-
and
insulin-induced
in-
sulin
resistance
was
related
to
the
levels
of
[Ca2+]i,
we
mea-
sured
[Ca2+]i
in
adipocytes
preincubated
under
the
same
con-
ditions
used
in
the
experiments
with
glucose
uptake
described
above
(Fig.
5).
Insulin
increased
[Ca2+]i
in
adipocytes
preincu-
bated
with
low
glucose
(1
mM)
in
the
presence
or
in
the
ab-
sence
of
insulin.
Basal
[Ca2+]i
was
not
increased
by
the
24-h
preincubation
with
10
mM
glucose,
but
in
these
adipocytes
acute
addition
of
insulin
failed
to
stimulate
[Ca2+]i.
Preincu-
bation
of
adipocytes
with
both
10
mM
glucose
and
10
ng/ml
Insulin
(10
ng/ml)
0
co
0a
n
0
0
0
(0
Li-
Glyburide
(2
paM)
M
Normals
M
Obese
Figure
4.
Effect
of
insulin
and
glyburide
upon
[Ca2"]i
in
adipocytes
obtained
from
six
normal
weight
controls
and
six
obese
women
after
overnight
fast.
Results
represent
mean±SEM;
P
<
0.01
(obese
vs.
normals).
insulin
raised
basal
[Ca2"]i
and
eliminated
the
effect
of
insulin
in
increasing
[Ca2"]i
(Fig.
6),
inducing
a
picture
similar
to
that
observed
in
adipocytes
obtained
during
euglycemic
clamp.
If
changes
in
[Ca2+]i
are
related
to
the
glucose-
and
insu-
lin-induced
insulin
resistance,
then
inhibiting
an
increase
in
[Ca2"]i
may
prevent
insulin
resistance.
Since
insulin
increases
[Ca2"]i
by
enhancing
Ca2"
influx
via
voltage-dependent
Ca2"
channels
(15),
we
incubated
adipocytes
as
described
above
in
the
presence
of
30
,uM
verapamil.
This
Ca"
channel
blocker
reduced
the
levels
of
[Ca2"]i
from
162±15
nM
to
99±13
nM
with
10
mM
glucose
alone
and
from
239±31
to
128±14
nM
with
glucose
and
insulin
(P
<
0.05),
and
restored
cellular
re-
sponsiveness
to
insulin
(Fig.
7),
suggesting
that
glucose-
and
insulin-induced
Ca"
influx
is
responsible
for
the
development
of
insulin
resistance.
Similar
restoration
of
adipocyte
response
to
insulin
was
achieved
in
preliminary
experiments
with
nifed-
ipine
(25
1AM)
or
cobalt
(0.5
mM)
(not
shown),
suggesting
that
a
blockage
of
Ca>2
influx
may
ameliorate
glucose-
and
insu-
lin-induced
insulin
resistance.
Discussion
The
present
data
directly
demonstrate
that
both
insulin
and
glyburide
increase
[Ca2+]i
in
human
adipocytes.
These
find-
24-HOUR
PREINCUBATION
1
mU
Glucose
I0
mM
Glucose
300
.
basal
Minsulin
5ng/ml
250
C
200
+
150
LJ100
50
00
10 0
10
PREINCUBATION
INSUUN
CONCENTRAlON
(ng/ml)
Figure
6.
Effect
of
24-h
preincubation
with
1
and
10
mM
glucose
(with
or
without
10
ng/ml
insulin)
on
basal
and
insulin-stimulated
[Ca2+]i
in
rat
adipocytes.
Results
represent
the
mean±SEM
of
five
to
eight
experiments.
1850
Draznin,
Sussman,
Eckel,
Kao,
Yost,
and
Sherman

0C
W
LU
,n
E
0
o
N."
P)
a
i
'
C
1.00
0.90
0.80
0.70
0.60
0.50
0.40
0.30
0.20
0.10
0.00
24-HOUR
PREINCUBATION
1
mM
Glucose
10
mM
Glucose
30
juM
veropomil
30,uM
verapomil
I
-
basal
J.
I
Minsulin
25ng/ml
0
10
0
10
PREINCUBATION
INSUUN
CONCENTRATION
(ng/ml)
Figure
7.
2-Deoxyglucose
uptake
in
adipocytes
preincubated
for
24
h
with
1
and
10
mM
glucose
(with
or
without
10
ng/ml
insulin)
in
the
presence
of
Ca2+
channel
blocker
verapamil
(30
MM).
Results
repre-
sent
the
mean±SEM
of
three
to
five
experiments.
A%,
%
stimulation.
ings
support
our
previous
observations
using
rat
adipocytes
(15),
where
both
insulin
and
glyburide
enhanced
calcium
in-
flux
via
voltage-dependent
calcium
channels.
In
this
study,
hyperinsulinemia
induced
by
3
and
6
h
of
insulin
(and
glucose)
infusion
not
only
increased
[Ca2+]i
in
adipocytes
obtained
from
normal
volunteers,
but
made
them
unresponsive
to
either
insulin
or
glyburide.
This
is
not
simply
a
desensitization
of
insulin
action,
because
the
effect
of
gly-
buride
was
also
eliminated.
The
diminished
responsiveness
of
adipocytes
to
insulin
was
also
manifested
by
the
reduced
insu-
lin-stimulated
glucose
uptake.
Although
the
coexistence
of
these
two
phenomena
does
not
prove
causality,
it
is
conceiv-
able
that
impaired
intracellular
Ca2'
homeostasis
contributes
to
the
diminished
cellular
responsiveness
to
insulin.
It
is
interesting
that
in
normal
subjects
hyperinsulinemia
of
relatively
short
duration
induced
the
same
degree
of
insulin
and
glyburide
resistance
as
that
seen
in
obese
individuals.
In-
deed,
obese
patients
demonstrated
higher
[Ca2+]i
in
the
basal
state
and
a
lack
of
response
to
either
insulin
or
glyburide
in
the
adipocytes
obtained
even
before
the
insulin
infusion.
During
the
course
of
the
insulin
infusion,
[Ca2+]i
remained
elevated
and
unresponsive
to
the
acute
influence
of
either
insulin
or
glyburide.
These
observations
may
indicate
that
obesity-asso-
ciated
high
levels
of
intracellular
free
Ca2'
produce
cellular
resistance
to
insulin
and
glyburide.
It
is
possible
that
these
represent
coexistent
abnormalities
that
are
not
necessarily
causally
related.
In
obese
patients,
mild
to
moderate
weight
loss
(8-12%)
failed
to
reduce
basal
[Ca2+]i
or
restore
adipocyte
responsive-
ness
to
insulin
and
glyburide.
The
lack
of
improvement
was
not
due
to
the
levels
of
fasting
insulinemia,
since
insulin
levels
in
the
obese
patients
were
not
different
from
controls
(6.5±1
MU/ml)
either
before
(7.2±1
uU/ml)
or
after
(6.8±2
uU/ml)
weight
reduction.
However,
plasma
insulin
levels
in
obese
pa-
tients
2
h
after
ingestion
of
75
g
glucose
were
significantly
higher
than
in
controls
(50±12
,uU/ml
before
and
39±12
,uU/ml
after
weight
loss
vs.
19±6
gU/ml
in
normals).
It
is
rational
to
postulate
that
postprandial
hyperinsulinemia
re-
mains
a
sufficient
stimulus
in
maintaining
high
levels
of
intra-
cellular
Ca2".
The
in
vitro
experiments
with
glucose-
and
insulin-induced
insulin
resistance
reported
in
this
communication
are
in
agreement
with
the
in
vivo
data.
In
both
cases,
exposure
of
adipocytes
to
high
glucose
and
insulin
concentrations
resulted
in
insulin
resistance
and
increased
levels
of
[Ca2"]i.
It
would
appear,
therefore,
that
if
hyperglycemia
and
hyperinsulinemia
induce
insulin
resistance,
this
insensitivity
must
develop
in
part
as
the
consequence
of
direct
action
of
glucose
and
insulin
on
peripheral
tissues
(in
this
case,
adipocytes).
It
has
been
previously
shown,
both
in
vivo
(22)
and
in
vitro
(20,
21,
23),
that
exposure
of
adipocytes
to
high
ambient
insu-
lin
levels
(particularly
in
the
presence
of
higher
levels
of
glu-
cose)
results
in
cellular
insensitivity
to
subsequent
insulin
stimulation.
The
decrease
in
cellular
sensitivity
and
respon-
siveness
to
insulin
were
attributed
to
insulin
receptor
and
postreceptor
defects,
with
the
latter
playing
a
predominant
role
(19-21,
23-26).
Our
data
suggest
that
sustained
high
levels
of
intracellular
Ca2l
may
contribute
to,
if
not
initiate,
the
postre-
ceptor
defects.
The
fact
that
hyperinsulinemia
and
high
levels
of
[Ca2+]i
induced
cellular
resistance
to
glyburide
suggests
that
[Ca2+]i
may
alter
the
cellular
response
to
multiple
agents.
However,
we
did
not
study
the
gamut
of
agents
stimulating
glucose
transport
in
adipocytes
and
therefore
cannot
draw
any
definitive
conclusions.
Insulin
has
been
shown
to
increase
Ca2+
influx
via
voltage-
dependent
Ca2+
channels
(15,
18),
and
its
effect
was
poten-
tiated
by
higher
ambient
glucose
concentrations
(15).
Insulin
can
also
affect
cellular
Ca2'
homeostasis
by
inhibiting
Na'-K'
ATPase
and
Ca2+-Mg2+ATPase
(for
review,
see
references
4
and
1
1).
The
fact
that
verapamil
restored
the
adipocytes'
re-
sponsiveness
to
insulin
does
not
necessarily
imply
that
Ca2+
influx
via
voltage-dependent
channels
is
the
only
mechanism
whereby
insulin
and
glucose
increase
[Ca2+]i.
The
loci
of
cel-
lular
insulin
and
glucose
action
in
increasing
and
maintaining
high
levels
of
[Ca2+]i
need
to
be
further
investigated.
The
precise
mechanism
whereby
higher
[Ca2+]i
induces
insulin
resistance
is
unknown.
We
observed
that
in
normal
rat
adipocytes,
insulin-stimulated
transport
of
2-deoxyglucose
was
inhibited
at
both
low
(with
calcium
channel
blockers)
and
high
(with
ionophore)
concentrations
of
intracellular
Ca2"
(18).
Similarly,
Bonne
et
al.
(27)
and
Taylor
et
al.
(28)
have
pre-
viously
shown
that
excessive
concentrations
of
calcium
(above
5
mM)
inhibited
the
effect
of
insulin
on
glucose
transport
in
isolated
adipocytes.
These
observations
are
consistent
with
the
possibility
that
persistently
high
[Ca2+Ji
may
contribute
to
the
overall
reduction
in
cellular
response.
The
influence
of
changes
in
[Ca2+]i
on
both
receptor
and
postreceptor
steps
of
insulin
action
has
not
been
studied.
Further
investigations
in
this
area
may
provide
new
insights
into
the
pathogenesis
of
insulin
resistance.
The
present
data
demonstrate
that
insulin
and
glyburide
are
capable
of
increasing
[Ca2+]i
in
isolated
human
adipocytes.
In
a
relatively
short
interval
of
time,
using
an
insulin
infusion,
it
is
possible
to
induce
resistance
to
the
action
of
insulin
and
glyburide
in
adipocytes
of
normal
subjects.
These
findings
also
suggest
that
under
certain
circumstances
(hyperinsulinemia
and/or
obesity)
increased
[Ca2+]i
may
be
a
factor
in
inducing
insulin
resistance.
Acknowledgments
The
authors
greatly
appreciate
the
excellent
secretarial
assistance
of
Gloria
Smith.
Cytosolic
Free
Calcium
Concentrations
and
Insulin
Resistance
1851

This
work
was
supported
by
the
Veterans
Administration
Medical
Research
Service,
grants
from
the
National
Institutes
of
Health
(AM-26356),
the
Upjohn
Company,
and
the
Mead-Johnson
Nutri-
tional
Division.
References
1.
Clausen,
T.,
J.
Elbrink,
and
B.
R.
Martin.
1974.
Insulin
control-
ling
calcium
distribution
in
muscle
and
fat
cells.
Acta
Endocrinol.
77:137-143.
2.
Kissebah,
H.
A.,
H.
Hope-Gill,
N.
Vydelingum,
B.
R.
Tullock,
P.
V.
Clarke,
and
T.
R.
Fraser.
1975.
Mode
of
insulin
action.
Lancet.
i:
144-147.
3.
Clausen,
T.
1980.
The
role
of
calcium
in
the
activation
of
the
glucose
transport
system.
Cell
Calcium.
1:311-325.
4.
Pershadsingh,
H.
A.,
and
J.
M.
McDonald.
1984.
Hormone-re-
ceptor
coupling
and
the
molecular
mechanism
of
insulin
action
in
the
adipocyte:
a
paradigm
for
Ca2"
homeostasis
in
the
initiation
of
the
insulin-induced
metabolic
cascade.
Cell
Calcium.
5:111-130.
5.
Bihler,
I.,
P.
Charles,
and
P.
C.
Sawh.
1980.
Effects
of
the
cal-
cium
ionophore
A-23187
on
the
regulation
of
sugar
transport
in
mus-
cle.
Cell
Calcium.
1:327-336.
6.
Klip,
A.
1984.
Is
intracellular
Ca2+
involved
in
insulin
stimula-
tion
of
sugar
transport?
fact
and
prejudice.
Can.
J.
Biochem.
62:1228-
1236.
7.
Klip,
A.,
G.
Li,
and
W.
J.
Logan.
1984.
Role
of
calcium
ions
in
insulin
action
on
hexose
transport
in
L6
muscle
cells.
Am.
J.
Physiol.
247:E297-E304.
8.
Desai,
K.
S.,
B.
Zinman,
G.
Steiner,
and
C.
H.
Hollenberg.
1978.
Effect
of
calcium
on
['25I]insulin
binding
to
rat
adipocytes.
Can.
J.
Biochem.
56:844-848.
9.
Plehwe,
W.
E.,
P.
F.
Williams,
I.
D.
Caterson,
L.
C.
Harrison,
and
J.
R.
Turtle.
1983.
Calcium-dependence
of
insulin
receptor
phos-
phorylation.
Biochem.
J.
214:361-366.
10.
Graves,
C.
B.,
R.
D.
Gale,
J.
P.
Laurino,
and
J.
M.
McDonald.
1976.
The
insulin
receptor
and
calmodulin:
calmodulin
enhances
in-
sulin-mediated
receptor
kinase
activity
and
insulin
stimulates
phos-
phorylation
of
calmodulin.
J.
Biol.
Chem.
261:10429-10438.
11.
McDonald,
J.
M.,
and
H.
A.
Pershadsingh.
1985.
The
role
of
calcium
in
the
transduction
of
insulin
action.
In
Molecular
Basis
of
Insulin
Action.
M.
Czech,
editor.
Plenum
Publishing
Corp.,
New
York.
103-117.
12.
McDonald,
J.
M.,
D.
E.
Bruns,
and
L.
Jarett.
1976.
Ability
of
insulin
to
increase
calcium
binding
by
adipocyte
plasma
membranes.
Proc.
Natl.
Acad.
Sci.
USA.
73:1542-1546.
13.
Goewert,
R.
R.,
N.
B.
Klaven,
and
J.
M.
McDonald.
1983.
Direct
effect
of
insulin
on
the
binding
of
calmodulin
to
rat
adipocyte
plasma
membranes.
J.
Biol.
Chem.
258:9995-9999.
14.
Pershadsingh,
H.
A.,
and
J.
M.
McDonald.
1979.
Direct
addi-
tion
of
insulin
inhibits
a
high
affinity
Ca2+-ATPase
in
isolated
adipo-
cyte
plasma
membrane.
Nature
(Lond.).
281:495-497.
15.
Draznin,
B.,
M.
Kao,
and
K.
E.
Sussman.
1987.
Insulin
and
glyburide
increase
cytosolic
free
Ca2+
concentration
in
isolated
rat
adipocytes.
Diabetes.
36:174-178.
16.
Sadur,
C.
N.,
and
R.
H.
Eckel.
1982.
Insulin
stimulation
of
adipose
tissue
lipoprotein
lypase:
use
of
euglycemic
change
technique.
J.
Clin.
Invest.
69:1119-1125.
17.
Rodbell,
M.
1964.
Metabolism
of
isolated
fat
cells.
J.
Biol.
Chem.
239:375-380.
18.
Draznin,
B.,
K.
E.
Sussman,
M.
Kao,
D.
Lewis,
and
N.
Sher-
man.
1987.
The
existence
of
an
optimal
range
of
cytosolic
free
calcium
for
insulin-stimulated
glucose
transport
in
rat
adipocytes.
J.
Biol.
Chem.
262:14385-14388.
19.
Marshall,
S.,
W.
T.
Garvey,
and
M.
Geller.
1984.
Primary
culture
of
isolated
adipocytes:
a
new
model
to
study
insulin
receptor
regulation
and
insulin
action.
J.
Bid.
Chem.
259:6376-6384.
20.
Garvey,
W.
T.,
J.
M.
Olefsky,
S.
Matthaei,
and
S.
Marshall.
1987.
Glucose
and
insulin
co-regulate
the
glucose
transport
system
in
primary
cultured
adipocytes:
a
new
mechanism
of
insulin
resistance.
J.
Bio.
Chem.
262:189-197.
21.
Sinha,
M.
K.,
L.
G.
Taylor,
W.
J.
Poriers,
E.
G.
Flickinger,
D.
Meelheim,
S.
Atkinson,
N.
S.
Sehgal,
and
J.
F.
Caro.
1987.
Long-term
effect
of
insulin
on
glucose
transport
and
insulin
binding
in
cultured
adipocytes
from normal
and
obese
humans
with
and
without
non-in-
sulin
dependent
diabetes.
J.
Clin.
Invest.
80:1073-1081.
22.
Mandarino,
L.,
B.
Baker,
R.
Rizza,
J.
Genest,
and
J.
Gerich.
1984.
Infusion
of
insulin
impairs
human
adipocyte
glucose
metabo-
lism
in
vitro
without
decreasing
adipocyte
receptor
binding.
Diabeto-
logia.
27:358-363.
23.
Kashiwagi,
M.,
A.
Verso,
J.
Andres,
B.
Vasquez,
G.
Reaven,
and
J.
E.
Foley.
1983.
In
vitro
insulin
resistance
of
human
adipocytes
isolated
from
subjects
with
non-insulin
dependent
diabetes
mellitus.
J.
Clin.
Invest.
72:1246-1254.
24.
Freidenberg,
G.,
R.
R.
Henry,
H.
H.
Klein,
D.
R.
Reichart,
and
J.
M.
Olefsky.
1987.
Decreased
kinase
activity
of
insulin
receptors
from
adipocytes
of
non-insulin
dependent
diabetic
subjects.
J.
Clin.
Invest.
79:240-250.
25.
Lonnroth,
P.,
M.
DiGiralamo,
and
U.
Smith.
1983.
Influence
of
ambient
glucose
and
insulin
concentrations
on
adipocyte
insulin
binding.
Metab.
Clin.
Exp.
32:609-614.
26.
Garvey,
W.
T.,
J.
M.
Olefsky,
and
S.
Marshall.
1986.
Insulin
induces
progressive
insulin
resistance
in
cultured
rat
adipocytes:
se-
quential
effects
at
receptor
and
multiple
post-receptor
sites.
Diabetes.
35:258-267.
27.
Bonne,
D.,
0.
Belhadj,
and
P.
Cohen.
1977.
Modulation
by
calcium
of
the
insulin
action.
Eur.
J.
Biochem.
75:101-105.
28.
Taylor,
W.
M.,
L.
Hau,
and
M.
L.
Halperin.
1979.
Stimulation
of
glucose
transport
in
rat
adipocytes
by
calcium.
Can.
J.
Biochem.
57:692-699.
1852
Draznin,
Sussman,
Eckel,
Kao,
Yost,
and Sherman