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Dissociation of in vitro sensitivities of glucose
transport and antilipolysis to insulin in NIDDM
HANNELE YKI-JARVINEN, KEIJI KUBO, JOANNA ZAWADZKI, STEPHEN LILLIOJA,
ANDREW YOUNG, WILLIAM ABBOTT, AND JAMES E. FOLEY
Clinical Diabetes and Nutrition Section, National Institutes
of
Health, National Institute
of
Diabetes and
Digestive and Kidney Diseases, Phoenix, Arizona 85016
YKI-JARVINEN, HANNELE, KEIJI KUBO, JOANNA ZAWADZKI,
STEPHEN LILLIOJA, ANDREW YOUNG, WILLIAM ABBOTT, AND
JAMES
E.
FOLEY.
Dissociation of in vitro sensitivities of glucose
transport and antilipolysis to insulin in NIDDM.
Am. J. Physiol.
253 (Endocrinol. Metab.
16): E300-E304, 1987.-It
is unclear
from previous studies whether qualitative or only quantitative
differences exist in insulin action in adipocytes obtained from
obese subjects with non-insulin-dependent diabetes mellitus
(NIDDM) when compared with equally obese nondiabetic sub-
jects. In addition, the role of changes in insulin binding as a
cause of insulin resistance in NIDDM is still controversial. We
compared the sensitivities of glucose transport and antilipolysis
to insulin and measured insulin binding in abdominal adipo-
cytes obtained from
45
obese nondiabetic (% fat,
41 k l), 25
obese diabetic (% fat, 40 t
l),
and
15
nonobese (% fat, 30 t
1)
female southwestern American Indians. Compared with the
nonobese group, the sensitivities of glucose transport and an-
tilipolysis were reduced in both the obese nondiabetic and obese
diabetic groups. Compared with the obese nondiabetic subjects,
the ED,, for stimulation of glucose transport was higher in the
obese patients with NIDDM
(171 t 38 vs. 92 t 10 pM, P <
0.005). In contrast, the EDSOs for antilipolysis were similar in
obese diabetic patients (32 t 6 PM) and obese nondiabetic
subjects (27 t 3 PM). No difference was found in insulin binding
in patients with NIDDM when compared with the equally obese
nondiabetic subjects. These data indicate
1)
the mechanism of
insulin resistance differs in NIDDM and obesity, and 2) the
selective loss of insulin sensitivity in NIDDM precludes
changes in insulin binding as a cause of insulin resistance in
this disorder.
insulin receptors; insulin resistance; diabetes
DECREASED SENSITIVITY t0
insulin’s effect on
glUCOSe
uptake both in vivo (7, 17, 26) and in vitro in adipocytes
(8, 15, 16, 25) is a characteristic metabolic abnormality
in subjects with non-insulin-dependent diabetes mellitus
(NIDDM) and/or obesity. The sensitivity of glucose up-
take to insulin in vivo (7) and glucose transport in
adipocytes in vitro (16) is more severely impaired in
subjects with NIDDM than in weight-matched obese or
nonobese nondiabetic individuals (7, 16). It is, however,
unknown whether the basic disturbance in insulin action
in NIDDM compared with obesity is qualitatively similar
and differs only in severity. One approach to resolve this
question is to define whether differences similar to those
found in insulin’s glucoregulatory action in NIDDM
compared with obesity also exist in other actions of
insulin.
In nondiabetic obese subjects, the insensitivity of glu-
cose transport to insulin in adipocytes has been a con-
sistent finding (8, 15, 16, 25). In contrast, the sensitivity
of antilipolysis to insulin has been reported to be either
normal (1,2,4,6,13,21) or reduced (15,25) in adipocytes
from obese nondiabetic subjects. In NIDDM, the sensi-
tivity of glucose transport to insulin is diminished (8,
16), whereas that of antilipolysis appears normal (5, 21).
Thus previous studies (1, 2, 4-6, 8, 13, 15, 16, 21, 25)
would predominantly support the view that the sensi-
tivity of antilipolysis to insulin is normal, whereas that
of glucose transport is decreased in both obesity and
NIDDM. However, no direct comparison of these proc-
esses has been performed to examine whether the mech-
anism of insulin resistance differs in obesity and
NIDDM.
Another unresolved issue regarding the mechanism of
insulin resistance in NIDDM is whether insulin obesity
to its receptors is reduced or not. Although the original
finding of reduced cellular insulin obesity in NIDDM by
Kolterman et al. (17) has not been confirmed by others
(5,16,21), the concept that a receptor defect plays a role
in the pathogenesis of insulin resistance in NIDDM is
still widely accepted (3, ‘17, 23).
In the present study, we compared the sensitivities of
glucose transport and antilipolysis to insulin and mea-
sured insulin obesity in abdominal adipocytes obtained
from weight-matched groups of obese nondiabetic and
diabetic (NIDDM) subjects and from a group of nonobese
nondiabetic subjects.
SUBJECTS AND METHODS
Subjects and experimental protocol. Eighty-five female
southwestern American Indians were admitted to the
Clinical Diabetes and Nutrition Center for study.
Twenty-five subjects had diabetes and 60 had normal
glucose tolerance according to National Diabetes Group
Criteria (22). The body mass index (BMI) in the diabetic
patients ranged from 28 to 61 kg/m’. The subjects with
normal glucose tolerance were divided into two groups
with a BMI either matched with the diabetic patients
(BMI 2 28 kg/ m2, obese groups) or with a BMI c 28 kg/
m2 (nonobese group). Clinical characteristics of the
groups are shown in Table 1. The subjects were untrained
and were not allowed to exercise while in the unit. Except
E300 0193-1849/87 $1.50 Copyright 0 1987 the American Physiological Society
GLUCOSE TRANSPORT AND LIPOLYSIS IN NIDDM AND OBESITY
E301
TABLE
1. Physical and biochemical
characteristics of study groups
Nonobese Obese Obese NIDDM
(15) (45)
(24)
Age, yr 24kl 2521 27tl
Body mass index, kg/m’ 24kl’ 37tl 39&l
Fat, % 30tl” 41tl 40*1
Fasting glucose, mg/dl 84t1 95tl 159+13$
Fasting insulin, pU/ml 22*3 43t3t 64&6
2-h Glucose, mg/dl 112t5 117t2 297*18$
2-h Insulin, pU/ml 130*20* 206t21 258246
Average fat cell size, nl 0.54t0.04* 0.8520.03 0.89t0.04
Values are means t SE; no. of subjects is in parentheses. Each
symbol denotes a significance at least at the 0.05 level for differences
between groups with the Bonferroni t test after analysis of variance.
* Nonobese group vs. obese nondiabetics and obese non-insulin-de-
pendent diabetes mellitus (NIDDM); t obese vs. the nonobese and
obese NIDDM; $ obese NIDDM vs. nonobese and obese NIDDM.
for diabetes and obesity, the subjects were in good health
as judged by history and physical examination and stand-
ard laboratory determinations. The patients consumed a
weight-maintaining diet containing (as percentage of
total calories) 50% carbohydrate, 30% fat, and 20% pro-
tein. After at least 3 days on this diet, an oral glucose
tolerance test was performed (22), and after 5 days
subcutaneous abdominal adipose tissue (5-15 g) was
removed from the lateral aspect of the hypogastrium as
previously described (16). Body fat content was deter-
mined by underwater weighing with correction for the
simultaneously measured residual lung volume (12). All
subjects gave written informed consent and the studies
were approved by the ethical committees of the National
Institutes of Health and the Indian Health Service and
by the Gila River Indian Community.
Measurement of glucose transport. Glucose transport
was determined using
D-[U-'4C]ghCOSe
in the presence
of a very low extracellular glucose concentration. Veri-
fication of this method including comparison with 3-0-
methyl-D-glucose has been reported previously (16).
Briefly, isolated adipocytes (2 % lipocrit) were incubated
in 500 ~1 of 5% albumin buffer in the presence of 0, 25,
50, 100, 200, 800, and 8,000 pM insulin and trace (300
nM) amounts of
D-[U-'4C]ghCOSc?.
The cell suspension
was incubated at 37°C for 1 h with continuous shaking
at 40 cycles/min. The incubation was terminated by the
oil method (II), and the amount of radioactivity associ-
ated with the adipocytes (as well as the total radioactivity
in the incubation medium) was determined by liquid
scintillation counting. The glucose transport rate was
expressed as the glucose clearance rate in femtoliters per
cell per second (vol in medium
x
cpm in cells/cpm in
medium) or per cell surface as attoliters per squared
micrometers per second.
Measurement of lipolysis. For measurement of basal
lipolysis, an aliquot of isolated cell suspension was added
to 5% albumin buffer (final ceil concentration, 5%; vol,
500 ~1) and incubated at 37°C for 2 h with continuous
shaking at
40
cycles/min. For measurement of insulin’s
antilipolytic effect, the cells were incubated in the pres-
ence of 25 nM L-isoproterenol and 12.5, 25, 50, 100, 200,
and 8,000 pM insulin. The incubation was terminated by
separating the cells from the medium by the oil method
(11).
In separate experiments it was shown that increas-
ing the cell concentration did not change the lipolysis
rate, thus indicating that the studies were done at satu-
rating adenosine concentrations. Insulin degradation,
which was determined by 10% trichloroacetic acid (TCA)
precipitability, during incubation was < 5% at all insulin
concentrations. Glycerol in the medium was determined
by an enzymatic assay, essentially as described previ-
ously by Wieland (29). Glycerol release was linear at
least 3 h. The rate of glycerol release was expressed per
cell (fmol l cell-’ . s-l) or per cell surface area [(lo-“‘) mol.
prns2. s-l].
Calculation of half-maximal transport rate. The con-
centration of insulin resulting in a half-maximal trans-
port rate (ED& was calculated for each individual sub-
ject from linear regression of transport rate vs. log of the
insulin concentration at 25, 50, 100, 200, and 800 pM. A
typical dose-response curve is shown in the study of
Kashiwagi et al. (16).
The concentration of insulin resulting in a half-maxi-
mal suppression of 25 nM isoproterenol-stimulated li-
polysis (ED50 for antilipolysis) was calculated from the
equation of linear regression of the percent of the lipo-
lytic rate (+insulin * lOO/-insulin) vs. the log of the
insulin concentrations at 12.5, 25, 60,
100,
and 200 pM.
Maximum suppression of lipolysis was defined as the
lipolytic rate at the inflection point (transition from
suppression of glycerol release to stimulation of glycerol
release) of the insulin dose-response curve. Because max-
imum suppression of lipolysis occurred at 100 or 200 pM,
the values at 100 or 200 pM were omitted if the values
at these concentrations were stimulatory. The correla-
tion coefficient of the lipolysis rate vs. the log insulin
concentration was >0.95 in all experiments.
Measurement of mono- 1251-[Tyr-A14]insulin binding.
Mono-1251- [Tyr-AL4]insulin binding was determined by
incubating a 300-~1 suspension (7%) of isolated adipo-
cytes in 5% albumin-HEPES buffer in the presence of
0.5 mg/ml bacitracin, 25 pM mono-‘““I- [Tyr-A14]insulin
(0.6-0.9 &i/pmol) and 0, 75, or 175 pM or 10 PM insulin
at 37°C for 1 h with constant shaking at 160 cycles/min.
The incubation was terminated with cold saline, and the
cells were separated from the medium by centrifugation
through oil (11). The cells were collected in a disposable
pipette tip and assayed for radioactivity on an auto-
gamma-counter (Packard). Radioactivity at concentra-
tions of 25, 100, and 200 pM insulin was corrected for
nonspecific binding by subtracting values obtained in the
presence of 10 PM of insulin. Bound insulin was calcu-
lated at each insulin concentration and expressed as the
amount of insulin bound per cell surface (fmol/pm2) or
as the volume cleared per cell [(incubation volume
x
specifically bound radioactivity)/ no. cells
x
free radio-
activity)].
Determination of cell size. Adipose cell size was deter-
mined by measuring osmium-fixed cells as described
previously (16).
Statistical methods. Analysis of data distribution and
statistical comparisons (analysis of variance followed by
Bonferroni’s
t
tests) were performed using standard pro-
E302
GLUCOSE TRANSPORT AND LIPOLYSIS IN NIDDM AND OBESITY
grams of the SAS Institute (Statistical Analysis System,
Cary, NC).
RESULTS
Glucose transport. Both basal and maximal glucose
transport rates (per cell and per cell surface area) were
reduced in the diabetic patients compared with weight-
matched nondiabetic subjects or nonobese subjects (Ta-
ble
2).
The sensitivity of glucose transport to insulin was
lower in both diabetic and nondiabetic obese groups
compared with nonobese subjects, and lower in diabetic
compared with nondiabetic weight-matched subjects
(Fig.
1A).
Lipolysis. The basal rate of glycerol release was ele-
vated in the diabetic patients compared with the weight-
matched subjects with normal glucose tolerance. The
EDsOs of insulin for antilipolysis were compared in the
diabetic patients and the weight-matched subjects with
normal glucose tolerance (Fig.
1A)
but in both groups
higher than in nonobese subjects (Table 2).
TABLE
2. Basal and maximum insulin-stimulated
glucose transport rate and basal- and isoproterenol-
stimulated rates
of
lipolysis in adipocytes
Nonobese
(1% Obese
(45) Obese DM
(25)
Glucose transport (per cell)
Basal, fl - cell-’ - s-’
Maximum, fl . cell-’ - s-’
Lipolysis
56+6
190+16
Basal, fmol - cell-’ - s-’
Maximum, fmol - cell-’ - s-’
Glucose transport (per cell
surface area)
0.08kO.01
0.6OkO.05
Basal, al - pm-’ - s-’
Maximum, al - prne2. 6’
Lipolysis
1.7k0.2
5.7kO.4
Basal, 10W21 mol. prns2 - s-’
2.3kO.3
Maximum,
10s21
mol. prns2 - s-’
17.3t1.0
56+5
152+15
0.15~0.01*
0.77kO.04
1.2t0.1*
3.3*0.3*
3.3Iko.3
16.6kO.5
41+4t
93+11t
0.21+0.03t
0.94+0.11$
0.9+0.lt
2.OkO.2 t
4.4+0.6$
19.7k2.3
Values are means + SE; no. of subjects is in parentheses. Each symbol
denotes a significance at least at the 0.05 level for differences between
groups with the Bonferroni t test after analysis of variance. * Obese vs.
nonobese and obese NIDDM; t obese NIDDM vs. nonobese and obese
NIDDM; $ NIDDM vs. nonobese.
ED50 OF INSULIN B BOUND INSULIN
- Gtocoso Tmmpor t Antilipolyri8 2SpM lnrulin
FIG. 1. A: sensitivities of glucose transport and antilipolysis to
insulin. B: insulin binding at tracer (25 PM) insulin concentration in
abnormal fat cells from patients with non-insulin-dependent diabetes
mellitus (n = 25; stippled bars) and obese nondiabetic subjects (n = 45;
open bars). * P < 0.001.
Insulin binding.
When expressed per cell surface area,
the amount of insulin bound at tracer insulin concentra-
tion
(25
pM) was similar in obese diabetic patients
(4.1
+ 0.2 fmol/pm’) and obese nondiabetic subjects (4.1 t -
0.1
fmol/pm2) but lower
(P <
0.05) in these two groups
than in the nonobese subjects (5.0 t
0.4
fmol/pm2). The
amount of insulin bound per cell was similar in in all
three groups (obese diabetics
194
t
13
pi/cell, obese
nondiabetics
188
t 8 pi/cell, and nonobese nondiabetics
167
t
14
pi/cell).
DISCUSSION
In the present study we found similar sensitivity of
antilipolysis but reduced sensitivity of glucose transport
to insulin in abdominal fat cells from patients with
NIDDM compared with equally obese nondiabetic sub-
jects. Insulin binding was comparable in both groups.
These data suggest that insulin sensitivity is not uni-
formly more severely impaired in NIDDM than in obe-
sity. Thus the mechanism of insulin resistance seems to
differ in these conditions. The finding of similar binding
in nondiabetic and diabetic obese subjects in the face of
the difference in the sensitivity of glucose transport
between the groups indicates that insulin resistance in
NIDDM cannot be explained by a decrease in insulin
binding.
The demonstration of a normal antilipolytic effect of
insulin in fat cells from subjects with NIDDM in the
present study is similar to that previously found by Arner
et al.
(1,
5). However, these investigators have also re-
peatedly found normal sensitivity of antilipolysis to in-
sulin in obese subjects
(1, 2, 4),
suggesting, contrary to
our present findings, that the mechanism of insulin
resistance is similar in obesity and in NIDDM. The
finding of a normal antilipolytic effect of Arner et al. in
adipocytes from obese subjects is in contrast to the
finding of reduced sensitivity of antilipolysis to insulin
in obese Caucasians
(25)
and Pima Indians (15). The
discrepant results are unlikely due to regional differences
in adipocyte metabolism, because both Arner et al.
(1)
and Pedersen et al. (25) used gluteal fat cells in their
studies. Whether methodological differences in the mea-
surement of lipolysis such as the use of saturating aden-
osine concentrations in the present studies and the use
of low adenosine concentrations in the studies of Arner
et al.
(1)
could explain the different results is unclear.
The “correctness” of the findings in these studies awaits
accurate quantification of in vivo rates of lipolysis in
obesity.
The finding of impaired insulin sensitivity of glucose
transport but normal sensitivity of antilipolysis to insu-
lin opens a new perspective to examine the significance
of possible changes in insulin binding as a cause of
impaired insulin action in NIDDM. The classic way of
evaluating the role of a change in insulin binding on
insulin action has been to determine whether binding is
normal or abnormal. However, interpretation of results
obtained by measuring “cell-associated” insulin binding,
as the assay usually is done
(1 l),
is complicated because
the cell-associated binding consists not only of receptor-
bound insulin in the cell membrane but also of internal-
GLUCOSE TRANSPORT AND LIPOLYSIS IN NIDDM AND OBESITY
E303
ized and degraded insulin (10,19). The amount of insulin
degraded and internalized varies depending on assay
conditions such as temperature and the concentration of
bacitracin (20, 24), and it may also be altered in subjects
with NIDDM (27, 28). An alternative way of studying
the role of insulin receptor changes as a cause of altered
insulin sensitivity is to examine the sensitivity of two
different pathways of insulin action. Regardless of the
observed receptor status, if the sensitivity of one pathway
is normal while that of the other is abnormal, the change
in sensitivity cannot be ascribed to a change in insulin
binding. If, on the other hand, the sensitivities of one or
more pathways of insulin action are similarly reduced,
then a change in insulin binding could be the cause of
reduced sensitivity. In the present study, we found loss
of sensitivity of glucose transport but not antilipolysis to
insulin in NIDDM and no change in insulin binding.
This result indicates selective impairment of insulin
action in subjects with NIDDM that cannot be ascribed
to a decrease in insulin binding.
In addition to the decreased sensitivity of glucose
transport to insulin, basal lipolysis was elevated and
basal as well as maximal glucose transport rates were
decreased in the diabetic patients. Regarding the patho-
physiological importance of these changes in the devel-
opment of insulin resistance in the human adipocyte,
there is so far no prospective data on the sequence by
which these alterations appear during transition from
normal to diabetic glucose tolerance. We recently studied
these parameters in a large group of non-diabetic non-
glucose-intolerant subjects with a range of normal glu-
cose tolerance (9). Interestingly, the subjects with highest
glycemic responses to oral glucose had similar rates of
basal lipolysis, basal and maximal glucose transport, and
similar sensitivity of antilipolysis to insulin than the
subjects with lowest glucose levels. However, the sensi-
tivity of glucose transport to insulin was reduced in the
subjects with highest glucose levels (9). These previous
cross-sectional data combined with the data from the
present study would suggest that the loss of sensitivity
of the glucose transport system to insulin is an early
event in the development of insulin resistance in human
adipocytes. The decrease in basal and maximal glucose
transport and the increase in basal lipolysis are late
events developing concomitantly with more marked fast-
ing hyperglycemia and/or relative insulin deficiency.
The authors acknowledge the excellent technical assistance of Pa-
mela Thuillez and Suzzane Moser and the nursing and dietary staffs
of the Phoenix Clinical Research Unit for their support.
This study was supported in part by grants from the Finnish Medical
Research Council (Academy of Finland, HY).
Received 6 October 1986; accepted in final form 14 April 1987. 23.
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