Reduction of liver mass due to malnutrition in rats.
Correlation with emaciation of animals and size of organs
not inserted in the portal system
D e p a r t m e n t o f H i s t o l o g y a n d E m b r i o l o g y o f t h e I n s t i t u t o d e C i e n c i a s B i o m e d i c a s o ft h e U n i v e r s i d a d e d e s a o P a u l o a n d I I S u r g i c a l D i v i s i o n , F a c u l d a d e d e M e d i c i n a d e
S o r o c a b a o f t h e P o n t i / i c i a U n i v e r s i d a d e C a t 6 l i c a . S o r o c a b a , B r a z i l
W e studied the effects of protein-energy m alnutrition on the liver m orphology of rats as com pared to anim al em aciation and to
reduction in size of the organs not irrigated by splanchnic blood such as kidneys and spleen. The anim als were divided into two
groups, one of them feda d lib itu m rate (N=10) and the other (N=14) receiving water but no food for 7 days, and the changes in anim al
weight, liver, kidney and spleen m ass were determ ined. DNA and the protein/DNA ratio, as well as hepatocyte size, were determ ined in liver tissue. The liver decreased in m ass (27.14% ) at a significantly higher proportion (p<O.05) when com pared to body em aciation (19.22% ). Sim ilar to the reduction in body weight, the m asses of kidneys and spleen were reduced by 18.68% and 24.28% , tively. The reduction in liver m ass occurred due to hypoplasia and atrophy, i.e., a decrease in hepatocyte num ber and size, respec-tively. W e conclude that there is a preferential consum ption of liver protein in protein-energy m alnutrition which is suggested to result from the additive action of the effects of overall consum ption of organic reserves due to m alnutrition proper and to the reduction of the hepatotrophic stim ulus.
UNITERM S: Atrophy. Liver. Glucagon. Insulin. Liver regeneration. M alnutrition. Starvation.
P
rotein-energym etabolic changesm alnutritionw hereby the organismis accom paniedattem ptsby to guarantee an energy supply to very im portant organs such as the heart and brain at the expense of fat and structural protein consum ption(lO ). T he result is em aciation or loss of body w eight including the protein m ass of som e internal organs, the Iiver in particular (1,2). In addition, there are concom itant changes in horm onalA d d re ss fo r co rre sp o n d e n ce : O so rio M ig u e l P a rra
R u a A ib i, 7 0
S a o P a u lo - S P - B ra sil- C E P 0 5 0 5 4 -0 1 0
hom eostasis that involve the availability of som e of the so called hepatotrophic factors (4,9,11,12,14,19,48,49). T he aim of the present investigation w as to study m orphom etric and biochem ical liver changes in m alnourished rats and to correlate them w ith the reduction in m ass of organs not irrigated by splanchnic blood, such as kidney and spleen, and not directly depending on portal hepatotrophic factors.
M ATERIAL AND M ETHODS
F em ale W istar rats w eighing approxim ately 200 g w ere identified individually and divided into tw o groups subm itted to the follow ing procedures:
PARRA, a.M .; HERNANDEZ-BLASQUEZ, F.J.H.; saUSA E SILVA, RAP. et aJ. - Reduction of liver m ass due to m alnutrition in rats. Correlation with em aciation of anim als and size of organs n n t in c o rto n inth o . n " rt~ 1 ~ \lC !tO n "l
183.9:t 1.4 6.25 :t 0.24 0.48 :t 0.02 1.26 :t 0.04
R E S U L T S
A 0.0339 :t 0.0012 0.0026:t 0.0000 0.0068:t 0.0001
B 0.0304 :t 0.0005 '0.0026:t 0.0000 0.0070:t 0.0001
T a b le 1
W e ig h ts (g ) o b s e rv e d in g ro u p A a n im a ls
n s K id n e y s
K id n e y s /b .w . S p le e n
n s S p le e n /b .w . L iv e r
G ro u p L iv e r/b .w .
p< 0.01
n s =n o t s ig n ific a n t
T able 3 reports the percent decrease in body and
organ w eight caused by m alnutrition in group B anim als.
T he decrease in liver w eight, w hen presented in this m anner, w as also proportional and significantly greater
than that of other organs in relation to em aciation. T a b le 2
O rg a n w e ig h t/b o d y w e ig h t (b .w .) ra tio o f c o n tro l a n im a ls (G ro u p A ) a n d o f e x p e rim e n ta l a n im a ls
(G ro u p 8 a t s a c rific e )
T he organ w eight/body w eight ratios listed in T able
2 show that, under the effect of prolonged starvation, the
liver decreased in w eight proportionally m ore than the
other organs, w ith a significant change in its relation to
body w eight. B o d y m a s s
M ean body w eight and w eights of the organs studied
for group A are presented in T able 1.
Statistical analysis: T he results are reported as m eans
and standard error of the m ean. G roups w ere com pared
by the Student t-test for tw o independent sam ples or for
paired sam ples w hen com parisons w ere m ade w ithin group
B . M ultiple com parisons w ere m ade by analysis of
variance and the discrim ination betw een groups w as
perform ed by the N ew m an-K euls test. T he level of
significance w as set at p< 0.05.
G roup A (control) - T en anim als w ere placed in a
single cage containing w ater and food ad libitum and left
there for 6 days.
A fter a 24 hour fast, the anim als w ere sacrificed by
ether inhalation on the 7th day and laparotom ized. T he
liver, spleen and both kidneys w ere rem oved, blotted dry
on filter paper and w eighed. Sm all fragm ents w ere
rem oved from the liver and fixed in 10% form alin for
m orphom etry, and a larger fragm ent w as im m ediately
. w eighed, identified and frozen for later biochem ical
determ inations.
G roup B - Fourteen anim als w ere placed in a single
cage and left there for 7 days w ith w ater ad libitum but no
food. A t the end of this period the anim als w ere sacrificed by ether inhalation, w eighed and subm itted to the sam e
procedures as described for the control group.
Indices relating anim al w eight to the w eight of the
organs studied (liver, spleen and kidneys) w ere extracted
from group A (control) and used to estim ate the initial
w eight of these organs in the living anim als of group B .
T he percent decrease in m ass of each of the organs studied w as then calculated at sacrifice using the form ula:
estim ated initial w eight - observed final w eight x 100
estim ated initial w eight
B iochem ical D N A m easurem ent w as perform ed after
extraction w ith trichloroacetic acid using the
diphenylam ine reaction (8,34) and liver tissue protein w as
m easured by the m ethod of L O W R Y (29). T he results of both m easurem ents are reported as concentrations (m g/g
liver) and as protein/D N A ratio.
T he total initial D N A and protein liver m asses of
group B anim als w ere calculated from liver w eight
estim ated at the beginning of the experim ent m ultiplied
by D N A and protein concentrations, respectively, in the
liver parenchym a of group A (control) anim als. T he total
final m asses w ere obtained by m ultiplying the final w eight of the livers of group B anim als by the respective D N A
and protein concentrations. T he data w ere then used to
calculate the difference betw een values at the initial tim e
and at the tim e after m alnutrition (percent variation).
T he liver fragm ents fixed in form alin w ere routinely
processed by paraffin em bedding, cutting into 6 flm
sections and staining w ith hem atoxylin-eosin (H E ).
C onsidering the num ber of nuclei per prefixed area as an indirect m easurem ent of cell size, i.e., the larger the num ber
of nuclei the sm aller the cell size (24) , the nuclei present
in 4 fields obtained at random from the liver of 5 anim als per group w ere counted. C ounts w ere done on fields
covered by a 0.0534 m m2
square obtained w ith a lO X grid eyepiece and a 40X objective attached to a binocular Z eiss
m icroscope. Fields containing portal spaces and
centrolobular veins w ere not considered.
s a o P a u lo M e d ic a l J o u rn a l/R P M 1 1 3 (3 ): 9 0 3 -9 0 9 ,1 9 9 5 P A R R A , a .M .; H E R N A N D E Z -B L A S Q U E Z , F .J .H .; S O U S A E S IL V A , R A P . e t a l. - R e d u c tio n o f liv e r m a s s d u e to m a ln u tritio n in ra ts . C o rre la tio n w ith e m a c ia tio n o f a n im a ls a n d s iz e o f o rg a n s
A 1 .9 4 9 :t 0 .0 6 8 1 3 6 .6 4 :t 5 .4 4 7 0 .5 0 :t 2 .8 3 1 7 9 .8 :t 6 .9 B 2 .1 0 6 :t 0 .0 6 9 1 2 3 .0 4 :t 4 .5 0 5 8 .8 9 :t 2 .1 5 2 4 1 .8 :t 5 .2 1 9 .2 2 :t 0 .4 5 * 2 7 .4 1 :t 1 .2 4 * 2 4 .2 8 :t 3 .5 4 1 8 .6 8 :t 1 .9 6 *p < O .0 5 b y a n a ly s is o f v a ria n c e a n d th e N e w m a n -K e u ls te s t.
T a b le 5
T o ta l D N A a n d p r o te in c o n te n ts (m g /liv e r ) b e fo r e a n d
a fte r m a ln u tr itio n in g r o u p B a n im a ls T a b le 3
E m a c ia tio n a n d d e c r e a s e in o r g a n w e ig h t in
g r o u p B a n im a ls (% )
Several substances such as nutritional elem ents and
horm ones, prim arily represented by insulin and glucagon,
have been reported to be factors contributing to the
hepatotrophic effect of portal blood responsible for the
m orphofunctional status of hepatocytes
(3,7,13,15,19,33,42,43,52). C arbohydrate, proteins and
am ino acids absorbed in the intestine, in addition to
contributing w ith energy and structural elem ents for cell
m aintenance and renew al (15), also act indirectly by
stim ulating the production of factors such as insulin itself
(17,20,21,22). T hus, it is to be expected that prolonged
nutritional deprivation and the consequent alterations in
m etabolism and horm onal hom eostasis w ill affect liver
trophism and the m aintenance of liver structure by
decreasing insulin availability (4,9,11,48), by changing the
insulin/glucagon m olar ratio (12,14,48,49), and by
reducing portal speed and flow (31). In m odels of
regenerative stim ulation by partial hepatectom y, it has been
dem onstrated that m alnutrition, and protein m alnutrition
in particular, acts as an inhibitory factor of hepatic
regeneration (6,41,46,47).
Food deprivation for a period of 7 days caused a
19.22% reduction in body w eight in the anim als used in
the present experim ent (T able 3), sim ilar to the 23.1 % value
obtained by A D D IS et al. (1) and the 18% value reported
by SK U L L M A N et al. (41). It is interesting to note that
kidney and spleen m asses w ere decreased in proportions
sim ilar to body w eight loss, but that the liver, an organ
inserted in the portal circuit, presented a proportionally
greater decrease, so that its relation to body w eight w as
significantly m odified (p< O .O O I) (T ables 2 and 3), a fact
also observed by SK U L L M A N et al. (41).
W ork by A D D IS et al. (1,2) has show n that the liver,
am ong all organs, loses greater am ounts of protein at a
faster rate, w ith a 20% loss w ithin as little as 2 days. W e
believe that the "preferential" consum ption of this protein
reserve is also due to another additional effect. It is know n
that shunt of the hepatopetal flow is accom panied by Iiver
atrophy (37,38), a fact attributed to the reduction of
hepatotrophic factors such as insulin (33,43). T hus, the
prolonged lack of food ingestion reducing the generation
of hepatotrophic factors, creates a condition of relative
insulin insufficiency (19), and possibly other factors,
sim ilar in its effects to that provoked by surgical shunt of
hepatopetal flow . T his fact is probably responsible for the
greater reduction in liver m ass in relation to body
em aciation and m ass of kidney and spleen, organs that do
not depend directly on the splanchnic blood, in an additive
D IS C U S S IO N
p < O .0 0 1
P e rc e n t v a ria tio n K id n e y s
p < O .0 1 S p le e n
P ro te in /D N A N u m b e r o f ra tio n u c le i/a re a
F in a l m a s s L iv e r
E s tim a te d in itia l m a s s
T able 5 reports the percent variation in total D N A
m ass and protein m ass per liver in the anim als of group B
betw een the beginning of the experim ent (estim ated
values) and the end of the 7-day period of starvation.
T a b le 4
L iv e r D N A a n d p r o te in (m g /g liv e r ) c o n c e n tr a tio n s
a n d p r o te in /D N A r a tio . N u m b e r o f h e p a to c y te n u c le i
p e r a r e a
n s n s n s = n o t s ig n ific a n t.
G ro u p D N A P ro te in c o n c e n tra tio n c o n c e n tra tio n
D N A 1 2 .3 9 2 :t 0 .3 3 3 * 9 .6 4 9 :t 0 .2 8 4 * -2 1 .8 1 :t 2 .3 0 + P ro te in 8 6 8 .3 3 :t 2 3 .3 5 ** 5 6 7 .1 3 :t 1 6 .6 8 ** -3 4 .6 4 :t 1 .1 2 + *p < O .0 0 1 ; **p < O .0 0 1 ; + p < O .0 0 1
T able 4 com pares the D N A and protein
concentrations and the protein/D N A ratio in the liver
parenchym a of the anim als of both groups. T he table also
com pares the num ber of nuclei/area, an indirect
m easurem ent of hepatocyte cell size.
E m a c ia tio n
P A R R A , a .M .; H E R N A N D E Z -B lA S Q U E Z , F .J .H .; s a u s A E S ilV A , R A P . e t a l. - R e d u c tio n o f
liv e r m a s s d u e to m a ln u tritio n in ra ts . C o rre la tio n w ith e m a c ia tio n o f a n im a ls a n d s iz e o f o rg a n s
nnl ino:::prtprl in Ihp nnrt::ll o:::vo:::lpm
action w ith respect to m ass reduction that w ould be
expected as a function of sim ple protein consum ption
caused by m alnutrition.
The role of glucagon is less clear since reports that
have related it to the trophic/regenerative stim ulus are
som etim es conflicting, now show ing that glucagon is
im portant for this process in rats and m ice (3,7,18,27),
now show ing that it plays no part in dogs (3,44,45).
H ow ever, several studies have show n that glucagon plays
at least an adjuvant role in the generation of the stim ulus
by m echanism s such as synergism w ith insulin (7,51),
m aintaining glucose supply in the presence of high insulin
levels (12), stim ulating am ino acid transport (16) or
increasing portal flow (28,32), actions that are certainly
altered by prolonged fasting.
The reduction in liver m ass seem s to have occurred
in tw o w ays, i.e., by a decrease in cell num ber (hypoplasia)
reflected by the decrease in liver size w ith sm all variations
in tissue D N A and protein concentrations (Table 4) or by
the significant reduction in total D N A and proteins (Table
5), and by the reduction in size of the rem aining
hepatocytes (atropy), as suggested by the increased num ber
of nuclei per area (p<O .O O I, Table 4), an indirect
m easurem ent of cell size, and by the significant decrease
(p<O .O O I) of the proteinlD N A ratio. A decrease in this
ratio and a sim ilar conclusion w ere reported by Y O U N G
et al. (53) in a study of low -calorie diets. The decrease in
hepatocyte size by reduced insulin availability has been
previously show n by STA R ZL et al. (42,43) in experim ents
w ith selective portal infusions.
It is also interesting to note that the reduction in
hepatic tissue protein per liver (-34.64% , Table 5) w as
significantly greater (p<O .O O1) than the reduction in D N A
(-21.81 % ), a value that roughly represents the num ber of
hepatocytes. Since this value is proportionally sim ilar to
the decrease in body w eight (19.22% ) and kidney w eight
(18.68% ), it m ay be assum ed that hypoplasia (decreased
num ber of cells) w as m ainly due to prolonged m alnutrition,
accom panying the process of general consum ption,
w hereas atrophy (reduction in cell size) w as due to a
reduction of the hepatotrophic stim ulus, i.e., to the reduced
availability of factors m ainly represented by insulin.
A lthough speculative, this hypothesis agrees w ith data
reported by SIEG EL et al. (39,40) w ho w ere able to
separate and individualize the hyperplastic and
hypertrophic stim uli, the latter being m ore related to the
availability of portal factors. In heterotopic transplants of
autologous liver lobes associated w ith Eck fistulae, these
investigators show ed an attenuation of the cell atrophy
process com pared to controls w ithout Eck fistulae, possibly
because portal factors such as insulin, w hich is largely
extracted during its first passage through the hepatic bed
(5,30,31), by not being consum ed as a function of the
portocaval shunt, m ay reach the autografts via the arteries
in a larger proportioR .
The process described here, i.e., hepatic hypoplasia
and atrophy, due to m alnutrition w ith the probable
participation of a decreased supply of hepatotrophic
factors, is in contrast to data observed in previous studies
in w hich the intraperitoneal (portal) infusion of additional
exogenous hepatotrophic factors caused an increase of as
m uch as 86% in the hepatic m ass of intact livers, w ith a
m ean value of 67.54% (35,36) reprodudng, in a w ay, the
increase in liver m ass observed w hen a liver is transplanted
into a recipient of larger size than the donor (23,25,26,50).
This show s that, from a m orphological view point, the liver
and its hepatocytes are extrem ely sensible to the variations
in trophic stim uli, and this fact m ay eventually be used in
therapeutic m anipulations of different Ii ver diseases.
Investigations on the functional aspects of these
situations are currently under w ay in our laboratory.
S ao P aulo M edical JournaU R P M 113(3): 903-909, 1995 P A R R A , a.M .;H E R N A N D E Z -B LA S Q U E Z , F .J.H .; S O U S A E S ILV A , R A P . et al. - R eduction of liver m ass due to m alnutrition in rats. C orrelation w ith em aciation of anim als and size of organs
RESUMO
O bjetivos: E s'tudar em ratos, a repercussao da desnutri9ao calorico-proteica na m orfologia hepatica, com parativam ente com 0 em agrecim ento dos anim ais e com a redu9ao do tam anho de orgaos nao irrigados por sangue esplancnico com o rins e ba90. M aterial e M tH odos: E m dois grupos de ratos, um alim entado "ad libitum " e outro sem alim ento.so com agua por 7 dias, foram detem inadas as m odifica90es no peso dos anim ais, nas m assas do ffgado, rins e ba90, protefna, D N A e rela9ao protefna/D N A no tecido hepatico e tam anho dos hepatocitos. R esultados: F oi observado que o ffgado dim inuiu de m assa (27,41 % ) em propor9ao significantem ente m aior (p< O ,05) que 0 em agrecim ento corporeo (19,22% ). R ins e ba90 tiveram suas m assas reduzidas em 18,68% e 24,28% , respectivam ente, sem elhante
a
dim inui9ao do peso corporeo. A redu9ao da m assa hepatica ocorreu por hipoplasia e atrofia, respectivam ente dim inui9ao no num ero e no tam anho dos hepatocitos. C onclusao: C oncluiu-se que existe um consum o preferencial de protefna hepatica na desnutri9ao calorico-proteica que poderia eventualm ente resultar da a9ao aditiva dos efeitos do consum o geral de reservas organicas pela desnutri9ao em si e da redu9ao do estfm ulo hepatotrofico.REFERENCES
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s h u n t in d o g s . L a n c e t,2 ( 7 9 4 7 ) : 1 2 4 1 - 1 2 4 2 , 1 9 7 5 .
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g lu c a g o n in f u s io n s o n liv e r m o r p h o lo g y a n d c e ll d iv is io n
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