Diabetologia 15,289-296 (1978)

D i a b e t o l o g i a
9 by Springer-Verlag 1978
Originals
Effects of Di et on the Cellular Insulin Binding
and the Insulin Sensitivity in Young Healthy Subjects
H. Beck-Nielsen, O. Pedersen, and N. Schwartz Sorensen
Medical Department III and Department of Clinical Chemistry, County Hospital, Tage Hansensgade, .2trhus, and Nutrition Laboratory,
Institute of Hygiene, University of Aarhus, Denmark
Summary. To ascertain whether the effects of diet on
glucose tolerance and insulin sensitivity are mediated
through changes of insulin receptors we have studied
insulin binding to monocytes in 24 young volunteers
(4 groups of 6) during 2 week periods of different
dietary regimens. In group 1 and 2 the subjects had
their usual diet plus 1000 kcal per day from sucrose
or fat, respectively. Group 3 had an isocaloric diet
with a low-sucrose content, while group 4 ate low-fat
high carbohydrate diets. Before change of diet the
total group of volunteers showed an inverse correla-
tion between insulin binding and average daily su-
crose intake (R = - 0.52, p < 0.01). An excessive
sucrose consumption (group 1) was associated with a
reduction both of insulin binding (p < 0.05) and
insulin sensitivity (p < 0.05). The changes of the two
variables were parallel (R = 0.95, p < 0.05). An
abundant fat intake (group 2) was also accompanied
by a decrease of insulin binding (p < 0.05). However,
insulin sensitivity was unaltered (p > 0.1). A rise of
insulin binding (p < 0.05) followed the isocaloric,
low-sucrose diet (group 3) whereas the insulin sen-
sitivity was unchanged (p > 0.1). After the isocaloric,
low-fat diet (group 4) no significant change of insulin
binding occurred (p > 0.1) whereas the insulin sen-
sitivity increased (p < 0.05). We conclude that diet
and especially the dietary sucrose content affect insu-
lin binding to human monocytes. Evidence is pre-
sented that changes of insulin sensitivity following
hyperalimentation of sucrose may be induced
through alterations of insulin receptors.
Key words: Insulin receptors, insulin sensitivity, diet,
sucrose, monocytes.
The composition and quantity of diet is of impor-
tance for glucose tolerance and insulin sensitivity. An
isocaloric diet high in polysaccharides but low in fat
increases glucose tolerance in normal subjects
whereas the opposite event occurs after intake of a
diet high in fat [1, 2, 3]. Intense hyperalimentation is
associated with an increase of the plasma insulin level
and a deterioration of glucose tolerance and insulin
sensitivity [4]. A high-sucrose diet leads to impaired
glucose tolerance and induces a concomitant rise in
the fasting plasma insulin concentration [5].
Patients with hyperinsulinism and impaired glu-
cose tolerance and insulin sensitivity are often found
to have decreased cellular insulin binding [6]. How-
ever, both insulin binding and insulin sensitivity rise
when the patients are treated with a hypocaloric diet
[6, 7]. Moreover, a positive correlation between insu-
lin binding and insulin sensitivity has been shown
both in normal and obese subjects [8, 9]. Cellular
insulin binding may thus be of importance in deter-
mining insulin sensitivity and changes in this binding
could explain, in part, the influence of diet on glucose
tolerance and insulin sensitivity. We have therefore
studied changes of insulin binding to monocytes and
changes of insulin sensitivity in young adults under
different dietary conditions.
Material and Methods
Normal Volunteers
28 young healthy persons, 7 males and 21 females, aged 23-33 yr,
within 80-120% of their ideal weight were studied. There was no
difference of ideal weight between males and females. All volun-
teers were nurses with the same degree of physical activity. Four
O012-186X/78/0015/0289/$01.60
290 H. Be c k- Ni e l s e n et al.: Ef f e c t s of Di e t on I ns ul i n Re c e p t o r s
Ta bl e 1. Cl i ni cal a n d e xpe r i me nt a l da t a i n 5 gr oups of y o u n g vol unt eer s , n = 6 i n gr oup 1 - 4 a n d n = 4 i n t he cont r ol g r o u p
Bo d y Fa s t i ng Fa s t i ng ki vl r r b Speci fi c
wei ght p l a s ma pl a s ma cel l b o u n d
i ns ul i n gl ucos e 10-~min -1 f r act i on c
(kg) (~t U/ ml ) ( mmol / l ) 10 -2
Group 1. Hyper cal or i c di et wi t h
hi gh s uc r os e c ont e nt
Be f or e s t udy 56. 6- +3. 0 6-+1 4. 6- +0. 2 4. 2_+0. 4 3. 3_+0. 2
Af t e r 7 days 57. 5_+2. 9 8_+1 5 . 1 a - 2.1_+0.2 a
Af t e r 14 da ys 58. 3- +3. 0 9___1 a 5.1__.0.1 a 3 . 3 +0 . 5 a 2.0_+0.1 a
Group 2. Hyper cal or i c di et wi t h
hi gh f at c ont e nt
Be f or e s t udy 61. 4- +4. 2 6-+1 5.0_+0.1 4 . 6 + 0 . 3 2.9_+0.3
Af t e r 7 da ys 62. 2_+4. 3 6_+1 5.1_+0.1 - 2 . 3 + 0 . 2 a
Af t e r 14 da ys 62. 8- +4. 3" 6-+1 5. 2-+0. 1 4. 8- +0. 6 2 . 3 + 0 . 2 a
Group 3. I socal or i c wi t h l ow
s ucr os e c ont e nt
Be f or e s t udy 57. 4_+3. 2 6_+1 5 . 3 + 0 . 2 4.4__+0.3 2. 8- +0. 3
Af t e r 7 da ys 58. 0- +2. 7 5 + 1 4.8_+0.1 - 2.6_+0.3
Af t e r 14 days 57. 0_+3. 0 4+-1 5.0_+0.1 4.1___0.6 3 . 8 + 0 . 4 a
Group 4. I socal or i c di et wi t h l ow
f at c ont e nt
Be f or e s t udy 6 8 . 5 +4 . 1 8+-1 5.2_+0.1 3.8___0.2 2 . 8 +0 . 3
Af t e r 7 days 68. 2_+3. 9 8+-1 4.6_+0.1 - - 2.5_+0.1
Af t e r 14 da ys 68. 0_+3. 4 8_+1 4.8_+0.1 4 . 6 a 3. 2_+0. 4
Controls
Be f or e s t udy 59. 5_+2. 8 5_+1 4. 6_+0. 2 - 3.3_+0.3
Af t e r 7 da ys 59. 3_+2. 6 - - - - 3.1_+0.5
Af t e r 14 da ys 59. 6- +2. 9 5+_1 4. 6_+0. 2 - - 3. 3_+0. 4
a Th e va l ue s ma r k e d " a " ar e si gni f i cant l y ( p < 0. 05) hi ghe r t h a n t he i ni t i al va l ue s
b kwr r r is t h e r a t e c ons t a nt f or t he fal l i n p l a s ma gl ucos e c onc e nt r a t i on a f t e r i nt r a ve nous i nj ect i on of 0. 05 U i nsul i n pe r kg b o d y wei ght
c Th e speci f i c cell b o u n d f r act i on is t he f r act i on of 125I-insulin b o u n d at t he t r acer i ns ul i n c onc e nt r a t i on
Ta bl e 2. Av e r a g e dai l y i nt a ke of f ood ( me a n -+ SEM) be f or e a n d dur i ng t he s t udy pe r i od i n 5 gr oups of y o u n g vol unt e e r s
Tot a l Tot a l c a r bo- Sucr os e Pr ot e i n Fa t
cal or i es hydr a t e s
( kcal / day) ( kcal / day) ( kcal / day) ( kcal / day) ( kcal / day)
Al c ohol
( kcal / day)
Group 1
Us u a l di et 1 9 8 6 + 1 7 2 6 9 7 + 6 2 168_+22 260_+32 9 2 5 + 9 8 106+_18
Hi gh s uc r os e di et 3247- +194 1847- +101 1147- +39 2 5 0 + 3 0 1 1 0 1 + 1 2 1 50- +18
Group 2
Us u a l di et 2380_+ 309 951_+113 268_+45 314_+35 1037_+106 79_+15
Hi gh f at di et 3756_+383 986_+119 290_+39 297_+34 2287_+184 86_+10
Group 3
Us u a l di et 1740_+128 640_+66 145_+45 235_+45 804_+55 61_+18
Lo w s uc r os e di et 1700_+67 454_+15 29_+10 327_+15 8 9 0 + 5 1 -
Group 4
Us u a l di et 2037+_150 655- +38 131+_13 314- +21 949-+ 80 116-+11
Lo w f at di et 2 0 5 0 + 1 6 9 1 0 9 3 + 9 1 196- +15 397_+20 560_+51 -
Controls
Us u a l di et 1 9 5 8 + 1 7 1 770- +82 1 9 0 + 2 8 3724- 32 7 4 4 + 6 6 72_+12
H. Beck-Nielsen et al.: Effects of Di et on Insulin Receptors 291
subjects (one in each study group) received contraceptive pills. No
ot her drugs were used. On days of examination the volunteers
were transported to the hospital by car to eliminate exercise before
blood sampling. Metabolic characteristics of the group are given in
Table 1.
with alpha naphthyl acetate esterase. The specific cell bound frac-
tion was adjusted to a standard monocyte concentration of 1.2
107/ml [16]. There was no significant change of monocyte concen-
tration in the five groups studied during the three week period
(p > 0.1). All binding studies were done in duplicate.
Prot ocol
The volunteers were randomly divided into 4 study groups, each
group comprising 6 subjects, and a control group of 4 subjects. A
trained dietitian instructed all attendants how to record food con-
sumption. Records were made at the end of each meal. The food
composition and the caloric content were estimated from dietary
tables [10]. Following 1 week of baseline evaluation, where the
volunteers were asked to continue their usual food intake, the
persons in group 1 and group 2 were put on hypercaloric diets
whereas the persons in group 3 and group 4 had isocaloric diets.
All continued their experimental diets for 2 weeks giving daily
records to the dietitian.
The subjects of group 1 were instructed to eat their normal
food plus 250 g pure sucrose/day giving 1000 extra kcal.
The subjects of group 2 were fed their normal diet with addi-
tion of cream to main meals giving a supplement of 1000 kcal per
day. The subjects of group 3 and group 4 were fed diets giving each
person the same daily energy supply as before the study. Group 3
ate a low-sucrose diet while group 4 had a low-fat diet. Table 2
gives the individual average daily intake of maj or constituents.
Before the diet was changed (baseline period) the following vari-
ables were measured:
Body weight, fasting plasma concentrations of glucose and
insulin, insulin binding to monocytes and intravenous insulin toler-
ance test (IVITT),
All measurements were repeated 1 week and again 2 weeks
after the change of diet, except I VI Tr , which was repeated only
after 2 weeks.
The controls continued their usual diet throughout the three
week period.
lnsulin: Plasma insulin concentration was measured by the
radioimmunoassay of Hedi ng [11].
Glucose: Plasma glucose concentration was measured using an o-
toluidine met hod [12].
Cell Binding Studies: One hundred and forty ml bl ood were drawn
from an antecubital vein at 8.00 a. m. after an overnight fast and
transferred to tubes containing EDTA (dipotassium salt). Mono-
nuclear leucocytes were isolated by gradient centrifugation [13].
The cells were washed twice and incubated in tris-HC1 buffer
(25 mmol/1, pH 8.0 at 15 ~ at a concentration of about 60
106/ml for 100rain at 15 ~ with 125 I-insulin (Novo Research
Institute, Copenhagen) at a concentration of 85 pmol/1 (0.5 ng/ml)
[13]. The specific activity of the tracer was 25- 30 ~tCi/~tg with
0. 1-0. 2 of iodine atoms per insulin molecule [14]. At this low
degree of iodination more than 95% of the iodoinsulin is mono-
iodinated [15].
For competition studies native insulin in increasing concentra-
tions was added to the incubation medium. At the end of the
incubation period cell bound and free insulin were separated by
centrifugation through silicone oil (relative density 1.04). "Specific
cell bound fraction" was defined as total cell bound fraction minus
non-specific cell bound fraction. Radioactivity which remained
bound in the presence of an excess of native insulin at 13 umol/1
was considered "non-specific". This fraction averaged 25% of
total binding. There was no significant difference between the non-
specific binding before and after dieting in the 4 groups studied.
The monocytes were identified in cytocentrifuged smears stained
Binding Analysis: The results of the binding studies are presented
in 4 ways:
1) The specific cell bound fraction (bound/total) of insulin at
the insulin tracer concentration, 85 pmol/l.
2) The specific cell bound fraction, plotted as a function of total
insulin concentration (competition curve).
3) Specific cell bound/ free insulin plotted as a function of
specific cell bound insulin (Scatchard plot) [17]. Maximal binding
capacity (Ro) was obtained by extrapolation of each curve to the
intercept on the abscissa.
4) The concentration of native insulin necessary to reduce the
specific cell bound fraction of 125 I-insulin 50% ("50% inhibi-
tion") is for comparative studies taken as an estimate for recept or
affinity (a low "50% inhibition" indicated a high receptor affinity
and vice versa).
Insulin Tolerance Test (IVITT): 0.05 U crystalline insulin per kg
body weight were injected intravenously. Blood samples for
plasma glucose were taken - 3 0 , - 15, 0, 5, 10, 15, 20, 25, 30, 35,
40 and 45 min after insulin injection. The insulin sensitivity was
expressed as the rate constant for plasma glucose disappearance
kiwwr = l n2/ T' h, in which TI/2 = half life time of the exponential
decline of the plasma glucose concentration between 5 min and 30
rain after insulin injection.
Statistical .Methods
Wilcoxon' s test for paired differences was employed for compari-
son of values before and after change of diet, while Spearman' s
coefficient of rank (R) was applied in correlation studies. Results
in figures and tables are given as mean values + SEM.
Results
I n t h e t o t a l g r o u p o f v o l u n t e e r s we h a v e s t u d i e d t h e
r e l a t i o n s h i p b e t we e n i n s u l i n b i n d i n g t o mo n o c y t e s
a n d f o o d i n t a k e b e f o r e t h e c h a n g e o f d i e t . T a b l e 3
s h o ws s i g n i f i c a n t l y n e g a t i v e c o r r e l a t i o n s b e t we e n
i n s u l i n b i n d i n g a n d t o t a l d a i l y f o o d c a l o r i e s , t o t a l c a r -
b o h y d r a t e i n t a k e a n d s u c r o s e i n t a k e , r e s p e c t i v e l y .
An i n s i g n i f i c a n t i n t e r r e l a t i o n s h i p wa s f o u n d b e t we e n
i n s u l i n b i n d i n g a n d f a t i n t a k e i n t h e t o t a l g r o u p o f
p a r t i c i p a n t s wh e r e a s t h e s a me 2 v a r i a b l e s we r e s i g-
n i f i c a n t l y n e g a t i v e l y c o r r e l a t e d i n f e ma l e s .
Effects of Experi ment al Diets
Group 1 (Hypercaloric, high-sucrose diet). The c e l l u -
l a r i n s u l i n b i n d i n g a t t r a c e r c o n c e n t r a t i o n wa s l o w-
e r e d a l r e a d y a f t e r 1 we e k a n d a f t e r 2 we e k s d i e t i n g
t h e i n s u l i n b i n d i n g wa s a p p r o x i ma t e l y 6 0 % o f t h e
i n i t i a l v a l u e ( p < 0 . 0 5 ) ( T a b l e 1). I t a p p e a r s f r o m t h e
S c a t c h a r d p l o t ( Fi g. 1 b ) t h a t a p o s s i b l e r e d u c t i o n o f
t h e r e c e p t o r c o n c e n t r a t i o n o c c u r r e d ( p < 0 . 1 ) a n d
f r o m t h e c o r r e s p o n d i n g c o mp e t i t i o n c u r v e ( Fi g. 1 a)
292 H. Beck-Nielsen et al.: Effects of Diet on Insulin Receptors
Table 3. Correlations between specific cell bound fraction and daily intake of total food, fat, carbohydrates and sucrose before change of
diet. The total number of subjects is only 27 as the food record of one female was incomplete
Total food Total fat Total carbohydrate Total sucrose
intake intake intake intake
(kcal/day) (kcal/day) (kcal/day) (kcal/day)
Cf ~ R = - 0 . 4 0 R = - - 0 . 2 5 R=- - 0 . 4 5 R = - 0 . 5 2
(n = 27) (p < 0.05) (p > 0.1) (p < 0.05) (p < 0.01)
Specific cell ~ R = - 0.60 R = - 0.42 R = - 0. 58 R = - 0.64
bound fraction (n = 20) (p < 0.02) (p < 0.05) (p < 0.02) (p < 0.01)
of 125I-insulin
Cf R = --0.30 R = --0.50 R = --0.21 R = - 0. 54
(n = 7) (p > 0.1) (p > 0.1) (p > 0.1) (p > 0.1
Specific cell bound
fraction
x 10-2
3.
2-
1-
a Total i nsul i n nmol/I
Bound/free i nsul i n
x 10-2
t o Before
3-1 9 After 7 days
ays
2-
I -
i00 zoo 300 e~0
b Insul i n bound pmol/I
Fig. 1. Insulin binding to monocytes in 6
normal subjects: effect of hypercaloric su-
crose feeding. Subjects were studied on their
usual diet and after 7 days and again after 14
days, respectively, when fed their usual diet
with addition of 1000 kcal sucrose per day.
Mononuclear leucocytes were incubated
with 125I-insulin (85 pmol/l) and native
insulin in increasing concentrations.
Radioactivity bound in the presence of
13 pmol/1 unlabelled insulin was called
nonspecific cell binding. Total binding
minus nonspecific binding gives the specific
cell bound fraction. Cell bound fractions
were corrected to a monocyte concentration
of 1.2 107/ml.
a Specific cell bound fraction of 125I-insulin
as a function of native insulin.
b Scatchard analysis of the binding data
from, a. Maximal binding capacity, Ro, was
obtained by extrapolation of each curve to
the abscissa
it is e vi de nt al so t ha t a p p a r e n t r e c e p t o r af f i ni t y
d e c r e a s e d ( p < 0. 05) si nce 5 0 %- i n h i b i t i o n of 125 I -
i nsul i n bi ndi ng wa s a c hi e ve d at 2. 0 n mo l / l i nsul i n i n
t he bas el i ne p e r i o d a n d at 2. 6 nmol / 1 2 we e ks af t er
t he s t ar t of t he hype r c a l or i c pe r i od. At t he e n d of t he
di et s t udy t he f as t i ng pl a s ma i nsul i n c o n c e n t r a t i o n
was i nc r e a s e d a b o u t 5 0 % ( p < 0. 05, Ta b l e 1)
wh e r e a s t he i nsul i n sensi t i vi t y (Fi g. 2 a n d Ta b l e 1)
was si gni f i cant l y i mpa i r e d ( p < 0. 05) . Be f o r e t he
c h a n g e of di et t he coef f i ci ent o f c or r e l a t i on ( R) f or
t he r e l a t i ons hi p b e t we e n t he i nsul i n sensi t i vi t y a nd
t he i nsul i n bi ndi ng at i nsul i n t r a c e r c o n c e n t r a t i o n was
0. 50, p > 0. 1 (Fi g. 3) whi l e t he R- v a l u e i nc r e a s e d t o
0. 95, p < 0. 05 (Fi g. 3) af t er h y p e r a l i me n t a t i o n of
s ucr os e. We i g h t gai n a v e r a g e d 1. 5 kg ( p < 0. 05) .
Group 2 (Hypercaloric, high-fat diet). The ef f ect of 2
we e k s i nc r e a s e d f at i nt a ke o n t he i nsul i n bi ndi ng t o
mo n o c y t e s is gi ven i n Ta b l e 1. Th e speci f i c cel l b o u n d
f r a c t i on at t r a c e r c o n c e n t r a t i o n was d e p r e s s e d 2 2 %
( p < 0. 05) . Th e c o mp e t i t i o n c ur ve (Fig. 4 a) i ndi -
c a t e d t ha t t hi s de c r e a s e was d u e t o a l owe r i ng of t he
r e c e p t o r af f i ni t y as 5 0 %- i n h i b i t i o n of t r a c e r b o u n d
i nsul i n was o b t a i n e d at 2. 0 nmol / 1 i nsul i n b e f o r e a nd
at 4. 0 nmol / 1 i nsul i n af t er t he hype r c a l or i c r e g i me n
( p < 0. 05) . Sc a t c ha r d anal ysi s of t he bi ndi ng d a t a
(Fi g. 4 b) r e v e a l e d a non- s i gni f i c a nt i ncr eas e ( p >
0. 1) of r e c e p t o r c onc e nt r a t i on. Co mp a r e d t o
bas el i ne, me a n f as t i ng p l a s ma l evel o f i nsul i n was
u n c h a n g e d ( Tabl e 1). Th e i nsul i n sensi t i vi t y was
u n a f f e c t e d b y t he hi gh f at i nt a ke (Fi g. 2 a n d Ta b l e 1).
Av e r a g e wei ght gai n was 1. 5 kg ( p < 0. 05) .
Group 3 (Isocaloric, low-sucrose, high-fat diet). On e
we e k af t er t he s t ar t of t hi s di et t he r e was n o si gni fi -
cant c h a n g e i n t he speci f i c cel l b o u n d f r a c t i on at
t r a c e r c o n c e n t r a t i o n ( Tabl e 1) wh e r e a s a si gni f i cant
ri se of t he s a me var i abl e o c c u r r e d at t he e nd of t he
s e c o n d we e k ( p < 0. 05) . Fi gur e 5 s hows t ha t t he ri se
i n i nsul i n bi ndi ng was c a u s e d b y a n i ncr eas e of r e c e p-
t or c o n c e n t r a t i o n ( p < 0. 05) wi t h o u t a n y c h a n g e of
r e c e p t o r af f i ni t y. Fa s t i ng p l a s ma i nsul i n c o n c e n t r a-
H. Beck-Nielsen et al.: Effects of Diet on Insulin Receptors 293
Plasma glucose
mmol/I Gr o u p 1
6
4
2 7
1
Plasma g~ucose Minutes
mmol/I Gr oup 3
3 -
2 -
o Before
;0
Plasma glucose
mmol/I Gr oup 2
4
1 4
1
0 1O 20 30 40 50
Minutes
Plasma glucose
ram01/! Gr o u p 4 3 ,
2 .
1,
50 10 20 30 40 50
Minutes Minutes
9 After
Fig. 2. Insulin tolerance tests for the 4 groups studied before and
14 days after change of diet (group 1: hypercaloric sucrose diet,
group 2: hypercaloric fat diet, group 3: isocaloric low sucrose diet
and group 4: isocaloric low fat diet). The plasma glucose concen-
tration is plotted as a function of time after IV injection of 0.05 U
insulin per kg body weight at time zero
Specific ceil bound
fraction
x 10-; o Before
R = 0.50, p>0.1
5.
9 After 14 days
R = 0.95, p<O.05
0
0 0
klvllT
Fig. 3. Correlation between k~wrr and the specific cell bound frac-
tion of 125I-insulin (85 pmol/l) for group 1 (hypercaloric sucrose
diet). For details see legend to Figure 1 and Figure 2
Specific cell bound
f ract i on
x 10 -2
3-
2-
1-
O,
o . 1 f . o l b 1 6 o
a Total i nsul i n nmo111
Bound/ f r ee i nsul i n
x l 0 -2
3. o Before
9 After 7 days
9 After 14 days
1
160 200 300 400
b I n s u l i n bound p mo l l l
Fig. 4. Insulin binding to monocytes from 6
normal subjects: effect of hypercaloric fat
feeding. Subjects were studied on their usual
diet and after 7 days and again after 14 days,
respectively, when fed their usual diet plus
1000 extra kcal per day arising from cream.
Experimental conditions as described in
legend to Figure i.
a Specific cell bound fraction of t25I-insulin
as a function of native insulin.
b Scatchard analysis of the binding data
derived from, a. Maximal binding capacity,
Ro, was obtained by extrapolation of each
curve to the abscissa
t i on, i nsul i n sensi t i vi t y a n d a v e r a g e b o d y we i ght we r e
u n c h a n g e d ( p > 0. 1) .
Group 4 (IsocaIoric, low-fat, high-polysaccharide
diet). No si gni f i cant c h a n g e i n i nsul i n bi ndi ng at
t r a c e r c o n c e n t r a t i o n ( p > 0. 1) was f o u n d af t er 2
we e k s di et i ng ( Ta bl e 1). No si gni f i cant c ha nge s of
r e c e p t o r n u mb e r ( p > 0. 1) or r e c e p t o r af f i ni t y ( p >
0. 1) o c c u r r e d (Fi g. 2). Th e f as t i ng p l a s ma i nsul i n
l evel r e ma i n e d c o n s t a n t wh e r e a s t he i nsul i n sensi t i v-
i t y ( Ta bl e 1 a n d Fi g. 2) i mp r o v e d si gni f i cant l y ( p <
0. 05) . Bo d y we i ght was ke pt c ons t a nt ( p > 0. 1) .
Controls. No si gni f i cant c ha nge s o c c u r r e d i n i nsul i n
bi ndi ng ( p > 0. 1) , i n f as t i ng p l a s ma c o n c e n t r a t i o n of
i nsul i n ( p > 0. 1) o r i n b o d y we i ght ( p > 0. 1) ( Ta bl e 1
a nd Fi g. 7).
D i s c u s s i o n
Th e r es ul t s o f t hi s r e p o r t d e mo n s t r a t e t ha t di et s wi t h
di f f er ent c o mp o s i t i o n a nd cal or i e c o n t e n t gi ven t o
he a l t hy y o u n g s ubj ect s l e a d t o a da pt i ve c ha nge s i n
cel l ul ar i nsul i n bi ndi ng a n d i n me t a b o l i c var i abl es.
294
Specific cell bound
f ract i on
x 10 -}
3-
2-
1-
i J
o . 1 1 . o i o 1 ~ o
a Total i nsul i n nmol / I
H. Beck-Nielsen et al.: Effects of Diet on Insulin Receptors
Bound/ f r ee i nsul i n
x '~
2-
o Before
9 Af t er 7 days
9 Af t er 14 days
100 200 300 400 500 600
I ns ul i n bound pmol / I
Fig. 5. Insulin binding to monocytes from 6
normal subjects: effect of isocaloric low-su-
crose feeding. Subjects were studied on their
usual diet and after 7 days and again after 14
days, respectively, when fed isocaloric but
low-sucrose diets. Experimental conditions
as described in legend to Figure 1.
a Specific cell bound fraction of 125I-insulin
as a function of native insulin.
b Scatchard analysis of the binding data
derived from, a. Maximal binding capacity,
Ro, was obtained by extrapolation of each
curve to the abscissa
Specific cell bound
f ract i on
x 10-2
3-
2-
1-
r
o . 1 f . o f o l b o
Total i nsul i n nmol / I
Bound/ f r ee i nsul i n
x 10-2
3-
o Before
9 Af t er 7 days
9 After 14 days
I n s u l i n bound pmol l l
Fig. 6. Insulin binding to monocytes from 6
normal subjects: effect of isocaloric low-fat
feeding. Subjects were studied on their usual
diets and after 7 days and again after 14
days, respectively, when fed isocaloric but
low-fat diets. Experimental conditions as
described in legend to Figure 1.
a Specific cell bound fraction of 125I-insulin
as a function of native insulin.
b Scatchard analysis of the binding data
derived from, a. Maximal binding capacity,
Ro, was obtained by extrapolation of each
curve to the abscissa
Specific cell bound
fraction
x10-2 j o Before
I ~ 9 After 7 days
3-
2-
1-
0. i 1.0 I i 0
a Total i nsul i n nmol I I
Bound/free i nsul i n
x 10 -2
3 -
1"
i00 200 300 400 500
b I nsul i n bound pmol I I
Fig. 7. Insulin binding to monocytes from 4
control subjects eating an unchanged diet.
a Specific cell bound fraction of 125I-insulin
as a function of native insulin.
b Scatchard analysis of the binding data
derived from, a. Maximal binding capacity,
Ro, was obtained by extrapolation of each
curve to the abscissa
H. Beck-Nielsen et al.: Effects of Diet on Insulin Receptors 295
During the baseline peri od insulin binding was
inversely correlated to the total daily food intake and
most not abl y to the sucrose content of the diet. The
effect of dietary sucrose on insulin binding to mono-
cytes was further support ed by the finding that inges-
tion of 250 g extra sucrose per day was followed by a
40% fall in insulin binding, whereas a reduction of
the sucrose consumpt i on was associated with a 30%
increase in insulin binding. On the ot her hand hyper-
caloric, high fat-containing diets depressed insulin
binding only about 20%. Isocaloric diets with low-fat
content did not affect insulin binding significantly.
However, the increase of dietary sucrose in group 4
(Table 2) may have diminished the expected rise in
cellular insulin binding of this group.
No changes of insulin binding occurred in con-
trols. Thus we conclude that the observed alterations
of insulin binding to monocyt es isolated from persons
who had their diet changed are real and neither
caused by methodological errors nor by day to day
variation.
Scatchard analysis of the competition studies
showed curvilinear plots (Figs. 1, 4, 5, 6). Whet her
this configuration of Scatchard plots is due to nega-
tive cooperativity among binding sites or the exist-
ence of more binding sites with different affinity and
capacity is currently discussed [18]. Therefore we
have applied "50% inhibition" (see ' met hods' ) as a
crude estimate of apparent recept or affinity.
Moreover, we have used the extrapolated intercept
on the x-axis of the Scatchard plot as an estimate of
the maximal binding capacity, Ro, because Ro is the
same no mat t er which of the two interpretations is
preferred. Anal ysed in this manner we found a
reduction of the binding affinity (p < 0.05) and
perhaps of the binding capacity (p < 0.1) following
hyperalimentation of sucrose while a rise of binding
capacity (p < 0.05) occurred after low-sucrose feed-
ing. Hypercal ori c fat feeding was associated with a
reduction of the binding affinity (p < 0.05). How-
ever, these conclusions are made with certain reser-
vations because of the difficulties in the interpreta-
tion of the Scatchard plots and the small study
groups.
Ip et al. [19] have report ed adaptive changes in
insulin receptors on adipocytes from rats fed a fat
diet. Cells from rats adapt ed to fat feeding bound less
insulin compared to those from rats fed a glucose
diet. However, Cushman and Salans [20] could not
detect any difference in insulin binding when adipo-
cytes from fat -fed rats were compared with those fed
a high-carbohydrate diet. Dahms et al. [21] have
explored the effect of high-fat and high-carbohydrate
but isocaloric diets on the insulin binding to adipo-
cytes in obese adults. No difference bet ween binding
to fat cells on either of the two diets were obtained.
The results of Dahms et al. may be compared to
those derived from group 4 in the present study in
which we found no significant change in insulin bind-
ing to monocyt es following a raised dietary carbohy-
drate: lipid ratio. Actually t here is no direct dis-
crepancy bet ween the results of Dahms et al. and
ours. On the ot her hand the results report ed by Ip et
al. [19] and Cushman and Salans [20] differ from
ours (group 1 and 2). In this context the difference in
protocol and diet should be remembered.
Our dat a suggest that the insulin recept or on
monocyt es is sensitive to changes of the diet. The
mechanism for this recept or modul at i on is unclear.
However, evidence is accumulating that insulin bind-
ing is inversely regulated by the ambient insulin con-
centration [22]. In the present study we found that
manipulations of the dietary sucrose (group 1 and 3)
were associated with inverse changes of the plasma
insulin concentration and the insulin binding to
monocyt es supporting the idea that sucrose might
influence the cellular insulin binding through the
plasma levels of insulin. However, no inverse changes
of the same two variables occurred in group 2 so we
conclude that plasma insulin under certain conditions
may be but one of many recept or regulatory factors.
Recent l y several studies have shown that insulin
binding and insulin sensitivity are related [8, 9].
These findings were confirmed in this study where a
positive correlation bet ween cellular insulin binding
and insulin sensitivity was demonst rabl e before the
change of diet ( R = 0.56, p < 0.01). We have esti-
mat ed changes bot h in insulin binding and insulin
sensitivity under dietary manipulations. In group 1
we found parallel changes in the two variables. The
correlation coefficient (R) increased from 0.50 to
0.95 during the diet peri od which indicates that the
changes in insulin sensitivity during sucrose feeding
can largely be explained by changes in insulin bind-
ing. Comparabl e changes in insulin binding and insu-
lin sensitivity occurred in group 4. Conversely, there
were no parallel changes in the groups with a high
lipid: carbohydrat e ratio (group 2 and 3), which is in
keeping with results obt ai ned during fasting [23]
where the two variables diverge. The changes in insu-
lin sensitivity in groups 2 and 3, therefore, must be
related to alterations of factors ot her than insulin
receptors e. g. intracellular enzymes. Our conclusions
assume that changes of insulin receptors on insulin
target tissues occur parallel to changes in monocytes.
Indeed, experimental dat a are available to support
9 .this assumption [24].
296 H. Beck-Nielsen et al.: Effects of Di et on Insulin Receptors
Acknowledgements. We are indebted to the dietitians Inger Marie
Jorgensen and Ul l a Vinther and to the laboratory workers
T. Skrumsager, L. Busch and L. Blak for skillful assistance, and
L. Thomsen for her careful preparation of the manuscript. The
study was supported by grants from Danish Medical Research
Council, Landsforeningen for Sukkersyges Fond, Nordisk Insulin
Fond and NOVO Fond.
References
1. Himsworth, H. P. : Dietetic factors determining the glucose
tolerance and sensitivity to insulin of healthy man. Clin. Sci.
Mol. Med. 2, 67-94 (1935)
2. Wilkerson, H. L. C., Hyman, H., Kaufman, M., McCuistion,
A. C. , O' S Francis, J.: Diagnostic evaluation of oral glucose
tolerance tests in nondiabetic subjects after various levels of
carbohydrate intake. N. Engl. J. Med. 262, 1047-1053 (1960)
3. Anderson, J. W. , Herman, R. H. : Effect of fasting, caloric
restriction, and refeeding on glucose tolerance of normal men.
Am. J. Clin. Nutr. 25, 41-52 (1972)
4. Sims, E. A. H., Danforth, E., Jr., Horton, E. S., Bray, G. A. ,
Glennon, J. A., Salans, L. B.: Endocrine and metabolic effects
of experimental obesity in man. Recent Prog. Horm. Res. 29,
457-494 (1973)
5. Szanto, S., Yudkin, J.: The effect of dietary sucrose on blood
lipids, serum insulin, platelet adhesiveness and body weight in
human volunteers. Postgrad. Med. J. 45, 602-607 (1969)
6. Olefsky, J. M. : The insulin receptor: Its role in insulin resist-
ance of obesity and diabetes (Review). Diabetes 25,
1154-1161 (1977)
7. Beck-Nielsen, H.: The pathogenic role of an insulin receptor
defect in diabetes mellitus of obese. Diabetes (in press)
8. Harrison, L. C., Martin, F. I. R., Melick, R. A.: Insulin recep-
tor binding in isolated fat cells and insulin sensitivity in obese.
J. Clin. Invest. 58, 1435-1441 (1976)
9. Beck-Nielsen, H., Pedersen, O.: Insulin receptors on mono-
cytes of young healthy persons correlated to glucose tolerance
and insulin sensitivity. Diabetologia 14, 159-163 (1978)
10. Helms, P.: Fodevaretabeller. Copenhagen: Akademisk Forlag
1973
11. Heding, L. G. : A simplified insulin radioimmunoassay
method. In: L. Donat o et al. (Ed.): Labelled proteins in tracer
studies 329-333. Brussels: Eurat om 1966
12. Feteris, W. A.: A serum glucose method without protein pre-
cipitation. Am. J. Med. Technol. 31, 17-21 (1965)
13. Pedersen, O., Beck-Nielsen, H.: A study of insulin receptors in
human mononuclear leucocytes. Act a Endocrinol. (Kbh.) 83,
556-564 (1976)
14. J~rgensen, K. H., Binder, C.: 125I-insulin as a tracer of insulin
in different chemical processes. In: L. Donat o et al. (Ed.):
Labelled proteins in tracer studies, 329-333. Brussels:
Eurat om 1966
15. Freychet, P., Roth, J., Neville, D. M. Jr.: Monoiodoinsulin:
demonstration of its biological activity and binding to fat cells
and liver membranes. Biochem. Biophys. Res. Commun. 43,
400~-08 (1966)
16. Beck-Nielsen, H., Pedersen, O., Kragballe, K., Sorensen,
N. S.: The monocyte as a model for the study of insulin recep-
tors in man. Diabetologia 13, 563-569 (1977)
17. S catchard, G.: The attractions of proteins for small molecules
and ions. Ann. N. Y. Acad. Sci. 51, 660-672 (1949)
18. Poller, R. J., Standaert, M. L., Haase, B. A.: Insulin binding to
the human lymphocyte receptor. J. Biol. Chem. 252,~
5828-5834 (1977)
19. Ip, C., Tepperman, H. M., Holohan, P., Tepperman, J.: Insulin
binding and insulin response of adipocytes from rats adapted
to fat feeding. J. Lipid Res. 17, 588-599 (1976)
20. Cushman, S. W. , Salans, L. B.: Effects of dietary composition
on insulin binding in isolated rat adipocytes (Abstract). Clin.
Res. 23, 317 (1975)
21. Dahms, W. T., Atkinson, R. L., Bray, G. A.: Insulin resistance
and the binding of insulin to adipocytes (Abstract). Clin. Res.
24, 116 (1976)
22. Gavin, J . R. , III, Roth, J., Neville, D. M. Jr., DeMeyt s, P.,
Buell, D. N.: Insulin-dependent regulation of insulin receptor
concentrations. A direct demonstration in cell culture. Proc.
Natl. Acad. Sei. USA 71, 84-88 (1974)
23. Olefsky, J. M. : Effects of fasting on insulin binding, glucose
transport, and glucose oxidation in isolated rat adipocytes:
relationship between insulin binding and insulin action. J. Clin.
Invest. 58, 1450-1460 (1977)
24. Olefsky, J. M. : Decreased insulin binding to adipocytes and
circulating monocytes from obese subjects. J. Clin. Invest. 57,
1165-1172 (1976)
Received: January 23, 1978, and in revised form." April 14, 1978
Dr. H. Beck-Nielsen
Medical Depart ment III
County Hospital
Tage Hansensgade
DK-8000 Aarhus C, Denmark