development of hypothalamic obesity in growing rats
TRANSCRIPT
Exp. Pathol. 1988; 33: 223-232 VEB Gustav Fischer Verlag Jena
Karl Marx University Leipzig, Institute of Pathological lliochemistry (Head: Prof. Dr. se. med. F. :\iULLER)
Development of hypothalamic obesity in growing ratsl)
By H. REMKE, A. WILSDORF and F. MULLER
With 7 figures
Address for correspondence: Dozent Dr. HARALD REMKE , Institnt fur Pathologische Biochemic der KMU, LiebigstraBe 16, Leipzig, DDR - 7010
Key wo r d s: obesity; hypothalamic obesity; adipocyte hypertrophy; monosodium glutamate; Na-L-glutamate; nucleus arcuatus ; eminentia media.na; growth hormone deficiency; fat accumulation; insulin; adipoblast.; adipose tissne; rat
Swnmary
Administration of monosodium gluatamate to neonate rats causes hypothalamic lesions in the region of the nucleus arcuatus and the eminentia mediana, followed by massive accumulation of triglycerides, diminished secretion of growth hormone, reduced body length and organ weights and diminished number of adipocytes (hypoplastic-hypertrophic obesity). Locomotor activity of obese animals is reduced by about 50 %. Food intake is increased by about 10 % during growth and development of obesity but decreased beneath the level of that in control animals in the stationary phase of obesity. Hyperinsulinemia coupled with insulin resistance develops in the stationary phase of obesity, i.e. when adipocyte diameter has reached approximately 100 f-tm. The effects of reduced secretion of growth hormone are eOllsidered to be a main factor of fat accumulation in this type of obesity.
Introduction
Animal obesity may develop after damage of paraventricular hypothalamic nuclei, on the basis of changed secretion pattern of insulin, catecholamines, sexual and thyroid hormones or enzyme defects in peripheral organ cells (SCLAFANI 1984; VASSELLI et al. 1983). Decreased expenditure of energy in relation to uptake is common to all these obesity types. A hypothalamic type of obesity has been c]lOsen for our investigations, which is inducible by administration of Na+ glutamate to neonate rats with an incidence of 100 % .
According to OLNEY (1969, 1971) and SERESS (1982) glutamate leads to lesions of nucleus arcnatus and the eminentia mediana followed by diminished sympathetic activity, deficiencies in the dopaminergic system and in the liberation of growth hormone releasing factor, sterility of animals and increase in vagotonia (KAWAKAMI et a1.1983; SARTIN et aI.1986). Na+ glutamate binds to membrane receptors of nerve cells of the above mentioned nuclei. In neonate rats affinity of glutamate to receptors is high (SANDERSON and MURPHY 1982) and because of low activities of glutamate metabolizing enzymes blood concentration of administered glutamate remains elevated for a longer time (AIROLDI et al. 1979). Binding of glutamatc is suggested to induce an increase influx of Ca2+ into nerve cells (PIN et al. 1984), thereby causing cellular lesions (LEIBOWITZ et al. 1983). This mechanism possibly includes a phosphorylation and inactivation of pyruvat dehydrogenase complex (SIEGHART 1981) leading to decreased glucose utilization and energy deficiency. This model of obesity offers the advantage that development and course of obesity may be studied in growing animals without superimposed secondary metabolic effects by induction of obesity in adult rats.
1) Dedicated to Prof. Dr. GUNTHER GEBHARDT, Head of the Center for Nutritional Seiences of tlw KarilVIarx University on the occasion of hi s 60th birthday.
223
In this paper glutamate induced obesity in rats is characterized by food intake, body growth, hormonal concentrations in the blood, lipid accumulation, cellular type of white adipose tissue and locomotor activity .
.Materials and lVlethods
Na+ L-gluta mate (2 mg per g b.w.) was administered subcutaneously to neonate Wi star rats daily from 1st to the 5th day after birth. 5-7 animals of a litter were treated with glutamate and the other animals of the litter served as controls. After weaning (21st day) control and glutamate treated animals were separated and breeding proceeded in separate cages for males and females. The animals had free access to food (pellets R-13, Schonwalde) and water. After an overnight fast animals were killed by decapitation and trunk blood was drawn for determination of metabolites and hormones. Insulin was measured radioimmunologically (Rotop-Radioimmun-Test Insulin, AdW der DDR), growth hormon e with a test kit for the human hormone (Rotop-Radioimmun-Test fiir HGH, AdW, DDR), using the partial crossreactivity against rat growth hormone.
The total body fat and protein content were estimated after boiling and homogenization of the whole body. Fat was analyzed after extraction with ether. Protein was estimated using the Kjeldahl method as the total N x 6.25.
As carcass weight is considered the body weight after decapitation, evisceration and removal of all abdominal fat depots. Volume and triglyceride content of adipocyt es were calculated by means of cell diameter by microscopical method (SJOSTROM et al. 1971). Oell number of the epididymal fat pads was calculat ed from the total lipid of tissue (according to FOLCn et al. 1957) and average cellular lipid content.
Locomotor activity of rats was estimated by an acoustic method according to HECKL-ENSZLI)f et al. (1984). For this purpose the animals were kept for 24 h in individual cages (in a dosed room, temperature 22 00). An acoustic sensor was installed at the wall of the cage, transferring mechanical vibrations into electrical signals. Frequencies and amplitudes of impulses were evaluated.
Statistical calculations of pair experiments were done by the WILCOXoN-test (WILCOXON and WILCOX 1964) and Student's t-test.
Results
Body and organ weights Glutamate obese rats (GOR) show diminished increase in body weight (fig. 1). At the
end of the growth phase body weight of GO R amounts to 83.5 % of that of control animals. The weight difference persists during further development. Male and female rats behave on principle in the same manner. Diminished growth also occurs for carcass and organ weights (table 1). Abdominal and especially epididymal fat mass corresponds to body fat mass. At 12 months of age body constituents reached the following percentual values related to body weight: fat 59.0/10.5; protein 9.8/22.8; mineral ash 1.8/4.5; and water 29.0/61.5 for GOR and control rats, respectively.
Table 1. Body and organ weights of male GOR and control animals. Breeding at 22 0 0 (x ± sx; x: p < 0.01 GOR vs. control animals)
a body weight (g) b carcass weight (g) e bfa· 100 d liver weight (g) e dfa· 100 f kidney weight (g) g ffa· 100 h tail length (em) I abdominal fat mass (g) k i fb· 100
11
GOR (days p.p.)
lJO
190 ± 8 (x) 12G ± 7 (x)
66 6.5 ± 0.2 (x) 3.5 1.7 ± 0.2 (x) 0.9
14.3 ± 0.8 (x) 8.0 ± 1.0 (x) G.3
11
224 Exp. Pathol. 33 (1988) 4
130
321 ± 11 (x) 206 ± 10 (x) 64
7.9 ± 0.9 (x) 2.5 2.2 ± 0.3 (x) 0.7
16.4 ± 1.0 (x) 24.8 ± 3.1 (x) 12.0 11
control animals (days p.p.)
60 130
258 ± 12 377 ± 17 178 ± 7 272 ±11
71 72.6 7.9 ± 1.1 10.2 ± 1.3 3.1 2.7 2.3 ± 0 9
.~ 2.8 ± 0.4 0.9 0.8
17.0 ± 0.5 20.1 ± 0.8 6.0 ± 0.7 10.1 ± 1.2 3.4 3.7
11 11
b.w.
3 4 5 6 8 9 10 11 12 13 14 15 month
Fig.l. Body weight of male GOR ( x ) and control animals (e). (x ± Sx; n = 11; x: p < 0.05; xx: p < 0.01).
0,18
0,16
0,14
0,12
0,10
ops 0,06
OP4
0,02
abdominal fat I carcass
(g/g1
4 (, 8 10 1Z lit 16 18 month
Fig. 2. Abdominal fat mass of male GOR ( x ) and control animals (e). (x ± sx; n = 15; xx: p < 0.01).
Fig. 2 shows the different development of abdominal fat mass. At the end of growth phase it amounts to 11 percent of carcass weight in male, 16 % in female GOR, resp. (control animals 3.5 % and 5 %, resp.).
Food intake
Fig. 3 demonstrates the diminished food intake for GOR in the dynamic and also in the stationary phase of obesity. Food intake is lowered by about 25% in GOR at the end of the growth phase. Related to body weight (specific food consumption) GOR, however, show a slight but significant hyperphagia between the 2nd and the 3rd month, corresponding to the dynamic phase of obesity. Conversely, during the further development the specific food consumption of GOR declines significantly beneath the level of that in control rats (fig. 4).
Exp. Pathol. 33 (1988) 4 225
food intake
Ig\ 30
20
10
, I ! ! ! I 1 ..
4 5 6 8 9 10 11
month
Fig. 3. Food intake of male GOR ( x ) and control animals (e). (x ± s,,; n = 15; x: p < 0.05; xx: p < 0.01).
160
120
80
40
food intake I b.w.
Ig/kg\
• • • xx .~ . xx. 1t
It)t,, •• •
X . )(
• )(
• Xx
•• xxx •••• l., •••• l ••
)C ,. xl<: )C X )( • x
~1~-L------~------~----~4------~~/~
month
Fig. 4. Specific food consumption of male GO R ( x ) and control animals (e).
Behaviour of lipid content at reduced environmental temperature and during starvation
Fig. 5 demonstrates the behaviour of abdominal fat by keeping animals at reduced environmental temperature (4°C). In control animals lipid accumulation is somewhat lower than at 22 °C. In contrast obese rats accumulate more lipid at 4°C than at 22°C. In the stationa,ry phase of obesity abdominal lipid content reaches approx. 21 % of carcass weight. Food intake was elevated in the cold environment for control animals and for GOR. In the latter it was obviously higher than in control rats. This cold induced hyperphagia (related to b.w.) in GOR is evident until the 3rd month. Thereafter the differences are no longer significant (table 2).
Decline of body weight and abdominal fat mass during starvation are shown in fig. 6. Abdominal lipid content was determined at the beginning and every two days in 4 animals of each group. The experiment was stopped when abdominal fat content reached 10 g per kg b.w.
226 Exp. Pathol. 33 (1988) 4
trJ
~
~
'"d
~
:or p ~ ~
~
Tab
le 2
. F
ood
inta
ke a
nd w
eigh
t ga
in i
n co
ld e
nvir
onm
ent
(4 °
C),
(x ±
sx)
age
(mon
th)
1 1.
5
GO
R
male
; n
= 1
0)
a fo
od i
nta
ke
(g/d
ay)
16
± 0
.9
24
± 1
.0
b bo
dy w
eig
ht
(g)
80
± 2
.8
140
± 5
.1
c aj
b (g
/kg)
20
0 17
1
con
tro
l a
nim
als
(m
ale
; 11
=
11
) d
food
in
tak
e (g
/day
) 17
.5 ±
1.0
23
.5 ±
0.9
e
bod
y w
eigh
t (g
) 98
±
4.8
16
5 ±
3.1
f
die
(g/k
g)
178
142
GO
R (
fem
ale
; n
= 1
0)
g fo
od i
ntak
e (g
/day
) 12
±
0.6
21
±
0.9
h
body
wei
ght
(g)
80
± 2
.3
125
± 1
.8
i g/
h (g
/kg)
15
0 16
8
con
tro
l a
nim
als
(fe
ma
le;
n =
10
) k
food
in
tak
e (g
/day
) 11
±
0.4
20
±
1.8
I
body
wei
ght
(g)
90
± 7
.6
130
± 2
.6
m k
/l
(g/k
g)
122
153
sign
ific
ance
s:
a vs
. d
n. s
. n.
s.
b vs
. e
p <
0.0
5 P
< 0
.01
g vs
. k
n. s
. Il
. S
.
h vs
. I
Il.
s.
n. s
.
~
from
1.5
mo
nth
: a
vs.
g,
p <
0.0
5; b
vs.
h,
P <
0.0
1; d
vs.
k,
P <
0.0
1; c
vs.
I, P
< 0
.001
!8
.... [\!)
[\
!)
-J
2 3
5
25.5
± 0
.9
27
± 1
.2
21
± 0
.9
210
± 5
.8
270
± 6
.2
320
± 9
.2
121
100
65
26.5
± 0
.8
28
± 0
.9
26.5
± 1
.4
250
± 4
.7
370
± 8
.6
400
± 9
.2
lOti
75
66
21
±1
.2
20.5
± 0
.9
18
±1.
1 15
0 ±
3.G
21
5 ±
4.3
21
5 ±
G.2
14
0 95
81
21
± 0
.7
HI
± 1
.1
18
± 2
.0
1 42
± 7
.0
230
± 2
.1
250
± 4
.3
147
82
72
n. s
. ll
. S
. P
< 0
.05
P <
0.0
1 P
< 0
.01
P <
0.0
1 Il
. S
. 11
. S
. P
< 0
.05
n. s
. p
< 0
.05
P <
0.0
1
0,24
0,20
0,1&
0,12
0,08
0,04
abdominal fat I carcass
(g ig )
t, 2
i, 4
t • 5
month
Fig. 5. Abdominal fat mass of male GOR ( x ) and control animals (.) breeding at reduefld environmental t emperature (4 °C). (x ± Sx; n = 11; x: p < 0.05; xx: p < 0.01).
100
80
&0 T
0.1&
0.12
0.08
OD4
b.w. (percent)
! I
4 6 fat I carcass
8 10 12 , .
14 day
day
Fig. 6. Decline of body. weight and abdominal fat mass during starvation. Experiment was performed with.rats 130 days p.p
These results demonstrate that storage of lipid in the adipose tissue can be mobilized nearly totally in GOR. Because of their larger fat depots GOR withstand a prolonged starvation time.
Plasma insulin and growth hormone Growth hormone concentration exhibits a typical age dependent behaviour (results not
given in this paper). In obese rats it is lower at each life time, already for 6 days p.p. (table 3). In contrast, insulin concentration is always nearly the same for GOR and control rats in the dynamic phase of obesity. However, hyperinsulinemia coupled with an increased blood glucose level develops in GOR during the stationary phase of obesity (table 4). This surprising result indicates that insulin has not a priming effect for development of this obesity. Fat accumulation seems to be more related to reduced release of growth hormone and to cellularity of white adipose tissue (V ASSELLI et al. 1983).
228 Exp. Pathol. 33 (1988) 4
Table 3. Plasma concentrations of growth hormone in GOR in relation to control animals (= 100%)
age GOR (days p.p.)
6 (n = 5) 38.6 30 (n = 15) 51.9
130 (n = 19) 53.9
Table 4. Comparison of insulin and glucose concentrations in GO R and control rats (male animals; x ± sx; x: p < 0.05; xx: p < 0.01; n.s.: not significant)
age Insulin (pmol/I) glucose (mmol/I) (days p.p.)
GOR control GOR control
45 252 ± 25 258 ± 28 5.1 ± 1.3 6.1 ± 1.2 n/sign. 10/n.s. 10 10/x 10 60 293 ± 52 268 ± 31 6.5 ± 0.9 6.5 ± 0.5 n/sign. 7/n.s. 7 11/n.s. 15 130 366 ± 41 266 ± 44 0.8 ± 0.7 6.6 ± 0.4 n/sign. 14/xx 14 18/xx 18 150 426 ± 86 249 ± 45 10.1 ± 1.2 6.3 ± 0.3 n/sign. 12/xx 12 12/xx 12 470 439 ± 186 216 ± 58 8.2 ± 1.5 6.9 ± 0.6 n/sign. 13/xx 13 12/xx 12
Table 5. Cellularity of epididymal white adipose tissue (x ± s; xx: p < 0.01; n.s.: not significant)
age wet weight ( day)
30 GO R 0.35 ± 0.03 n/sign. 7/n.s. 30 control 0.36 ± 0.06 n 7
60 GO R 2.8 ± 0.08 n/sign. 10/xx 60 control 1.9 ± 0.10 n 10
130 GOR 9.6 ± 1.9 n/sign. (n) 19/xx 130 control 5.0 ± 0.8 n (Ii.) 19
470 GOR 12.5 ± 2.4 n/sign. (n) 9/xx 470 control 8.7 ± 2.2 n (n) 9
Ce llularit y of whit e adipose tissue
adipocyte diameter Cum)
54.2 ± 17.9 7/n.s. 49.3 ± 7.7 7
89.7 ± 10.0 10/xx 65.2 ± 7.1 10
142.4 ± 17.1 19/xx 85.5 ± 11.4 19
144.5 ± 16.9 9/xx 93.3 ± 11.8 9
adipocyte per fat pad ( x 106)
6.3 ± 1.0 10/xx 10.9 ± 2.3 10
5.8 ± 1.1 19/xx 12.4 ± 1.1 19
7.9 ± 3.1 9/xx 19.2 ± 5.7 9
Table 5 demonstrates that fat cell number in white adipose tissue is reduced in GOR. This holds for cell number per g of differently localized white adipose tissue and also if calculated for total epididymal fat pad. The hypoplasia of white adipose tissue in GOR is in coincidence with diminished growth hormone in blood, reduced body length and organ weights. Cell number in epid idymal adipose tissue remains nearly constant at a low level during growth phase of GOR. Mass increase in tissue only occurs by enlargement of cell volume.
Lipid accumulation and the typical cellularity in GOR also occurs under restricted food consumption by intake of 2/3 of ad libitum consumption.
15 Exp. Pathol. 33 (1988) 4 229
400 impulse / h
300 GOR
200
100 ~ P-6 12 18 24 6
400
300 control
JJJfLillm 200
100 lh i
6 12 18 24 6 a
200 impulse / h
150 GOR
100
50 110 I rlb--rn p-D-- "'"p 6 12 18 24 6
200
150 control
JUJlllIhTh 100
50
P .nJJ 12 1B 24 6 b
Fig. 7. Locomotor activity of male GOR and control animals a: 60 days p.p.; b: 130 days p.p. (x; n = 6).
Locomotor activity GOR exhibit reduced physical activity. Fig. 7 shows the frequency and diurnal pattern
of electrical impulses generated by the noise of movements of rats. The product of impulse height and frequency is lower by approximately 50 % in GO R. Also the diurnal rhythm is less marked. Because locomotor activity is diminished as well in 60-day-old GOR (at the beginning of fat accumulation) as in 130-day-old ones, locomotor inactivity cannot be the result of fat accumulation. Conversely it seems to be a direct effect of the hypothalamic lesion and may contribute to development of obesity by lowering energy consuming reactions.
Discussion
Glutamate administration induces lesions of well known parts in the paraventricular hypothalamic region in neonate rats, leading to phenotypic obesity and diminished locomotor activity. By an excess accumulation of triglycerides during growth this type is characterized as a true model of obesity (V ASSELLI et al. 1983). Sympathetic neurons in the nucleus arcuatus and the eminentia mediana are parts of the central control system for secretion of
230 Exp. Pathol. 33 (1988) 4
growth hormone (Acs et al. 1982; BLOCH et al. 1984) and gonadotropins (NEMEROFF et al. 1981; DALKIN et aI.1985). The injury of these somatoliberin producing neurons explains the diminished plasma concentration of growth hormone and further the retardation of body growth, reduced body length and organ masses in GOR. Therefore body mass alone will not be a certain mark for characterizing obesity development. Abdominal fat mass is more suitable for this purpose (V ASSELLI et al. 1983).
Quantitative evaluation of fat cell number and of adipocyte diameters reveals that adipose tissue is of hypertrophic and hypoplastic type. Hypoplasia of white adipose tissue as a peculiarity of GOR is obviously the result of diminished plasma growth hormone existing in that period when in control animals an increase in fat cell number takes place by proliferation of adipoblasts. In GOR only 46.7 of adipocytes compared to control rats have been found.
Other hypothalamic types of obesity induced by electrolytic destruction, knife cuts or goldthioglucose administration are immediately after injury of VMK connected with hyperinsulinemia (HANSEN et al. 1983; BRAY et al. 1981). Conversely glutamate induced hypothalamic lesion does not result in hyperinsulinemia during the developmental dynamic phase of obesity. According to our results plasma concentration of insulin does not increase until fat accumulation reaches a stationary phase. This suggests elevation of plasma glucose level and hyperinsulinemia to be a consequence of obesity probably by insulin resistance (FELlG 1984). The typical changes of cellularity in white adipose tissue of GOR correspond to those in children with diminished secretion of growth hormone (BOHLES 1980). Also in adult humans (esp. in cases of hypertrophic obesity) diminished concentrations of growth hormone have been found (CROCKFORD and SALMON 1970; WILLIAMS et al. 1984).
These conditions correlate with typical metabolic deficiencies, e.g. incidence of coronary disease (BJORNTORP 1985) and diabetes mellitus type II (FELIG 1984). In GOR diabetes mellitus develops in a very early lifespan and therefore this model can be used to follow up some pathogenetic aspects connected with obesity.
Obviously a depressed activity of sympathetic nervous system occurs and that is responsible for diminished food induced thermogenesis in GOR (unpublished results). But there are no proofs indicating a permanent defect in satiety controlled by the nucleus ventromedialis.
Energy intake is elevated only in the growth phase to a minor extent but declines in the stationary phase of obesity beneath the level of control rats. Also the effective adaption of GOR to cold environment does not agree with deficiencies of other parts of the sympathetic nervous system out of nucleus arcuatus and eminentia mediana.
Because obesity nearly under normophagic conditions energy expenditure, e.g. food induced thermogenesis and/or locomotor activity, must be restricted. Results on locomotor activity of GOR have been discussed controversially until now (ARAUJO and MAYER 1973; TAKASAKI et al. 1979) and its role in and contribution to development of obesity remain unclear. According to our results the low locomotor activity and also the less marked circadian rhythm may not be a consequence of obesity in GOR. More likely it belongs to the picture of the glutamate induced hypothalamic lesion and possibly is related to diminished sympathicotone (KOLAR and JANSKY 1984). In order to clarify the role of catecholamines, norepinephrine mediated reactions are currently under investigation.
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(Received February 10, 1987; Revised June 4, 1987)
232 Exp. Pathol. 33 (1988) 4