Reduction of liver mass due to malnutrition in rats . Correlation with emaciation of animals and size of organs not inserted in the portal system

We studied the effects of protein-energy malnutrition on the liver morphology of rats as compared to animal emaciation and to reduction in size of the organs not irrigated by splanchnic blood such as kidneys and spleen. The animals were divided into two groups, one of them fed ad libitum rate (N=10) and the other (N=14) receiving water but no food for 7 days, and the changes in animal weight, liver, kidney and spleen mass were determined. DNA and the protein/DNA ratio, as well as hepatocyte size, were determined in liver tissue. The liver decreased in mass (27.14%) at a significantly higher proportion (p<O.05) when compared to body emaciation (19.22%). Similar to the reduction in body weight, the masses of kidneys and spleen were reduced by 18.68% and 24.28%, respectively. The reduction in liver mass occurred due to hypoplasia and atrophy, i.e., a decrease in hepatocyte number and size, respectively. We conclude that there is a preferential consumption of liver protein in protein-energy malnutrition which is suggested to result from the additive action of the effects of overall consumption of organic reserves due to malnutrition proper and to the reduction of the hepatotrophic stimulus.


P
rotein-energy malnutrition is accompanied by metabolic changes whereby the organism attempts to guarantee an energy supply to very important organs such as the heart and brain at the expense of fat and structural protein consumption(lO).
The result is emaciation or loss of body weight including the protein mass of some internal organs, the Iiver in particular (1,2).In addition, there are concomitant changes in hormonal Address for correspondence: Osorio Miguel Parra Rua Aibi, 70 Sao Paulo -SP -Brasil-CEP 05054-010 homeostasis that involve the availability of some of the so called hepatotrophic factors (4,9,11,12,14,19,48,49).
The aim of the present investigation was to study morphometric and biochemical liver changes in malnourished rats and to correlate them with the reduction in mass of organs not irrigated by splanchnic blood, such as kidney and spleen, and not directly depending on portal hepatotrophic factors.

MATERIAL AND METHODS
Female Wistar rats weighing approximately 200 g were identified individually and divided into two groups submitted to the following procedures: 904.

RESULTS
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  The decrease in liver weight, when presented in this manner, was also proportional and significantly greater than that of other organs in relation to emaciation.

Table 2
Organ weight/body weight (b.w.) ratio of control animals (Group A) and of experimental animals (Group 8 at sacrifice) The organ weight/body weight ratios listed in Table 2 show that, under the effect of prolonged starvation, the liver decreased in weight proportionally more than the other organs, with a significant change in its relation to body weight.

Body mass
Mean body weight and weights of the organs studied for group A are presented in Table 1.

Statistical analysis:
The results are reported as means and standard error of the mean.Groups were compared by the Student t-test for two independent samples or for paired samples when comparisons were made within group B. Multiple comparisons were made by analysis of variance and the discrimination between groups was performed by the Newman-Keuls test.The level of significance was set at p<0.05.
Group A (control) -Ten animals were placed in a single cage containing water and food ad libitum and left there for 6 days.
After a 24 hour fast, the animals were sacrificed by ether inhalation on the 7th day and laparotomized.The liver, spleen and both kidneys were removed, blotted dry on filter paper and weighed.Small fragments were removed from the liver and fixed in 10% formalin for morphometry, and a larger fragment was immediately .weighed, identified and frozen for later biochemical determinations.
Group B -Fourteen animals were placed in a single cage and left there for 7 days with water ad libitum but no food.At the end of this period the animals were sacrificed by ether inhalation, weighed and submitted to the same procedures as described for the control group.
Indices relating animal weight to the weight of the organs studied (liver, spleen and kidneys) were extracted from group A (control) and used to estimate the initial weight of these organs in the living animals of group B. The percent decrease in mass of each of the organs studied was then calculated at sacrifice using the formula: estimated initial weight -observed final weight x 100 estimated initial weight Biochemical DNA measurement was performed after extraction with trichloroacetic acid using the diphenylamine reaction (8,34) and liver tissue protein was measured by the method of LOWRY (29).The results of both measurements are reported as concentrations (mg/g liver) and as protein/DNA ratio.
The total initial DNA and protein liver masses of group B animals were calculated from liver weight estimated at the beginning of the experiment multiplied by DNA and protein concentrations, respectively, in the liver parenchyma of group A (control) animals.The total final masses were obtained by multiplying the final weight of the livers of group B animals by the respective DNA and protein concentrations.The data were then used to calculate the difference between values at the initial time and at the time after malnutrition (percent variation).
The liver fragments fixed in formalin were routinely processed by paraffin embedding, cutting into 6 flm sections and staining with hematoxylin-eosin (HE).Considering the number of nuclei per prefixed area as an indirect measurement of cell size, i.e., the larger the number of nuclei the smaller the cell size (24) , the nuclei present in 4 fields obtained at random from the liver of 5 animals per group were counted.Counts were done on fields covered by a 0.0534 mm 2 square obtained with a lOX grid eyepiece and a 40X objective attached to a binocular Zeiss microscope.
Fields containing portal spaces and centrolobular veins were not considered.Several substances such as nutritional elements and hormones, primarily represented by insulin and glucagon, have been reported to be factors contributing to the hepatotrophic effect of portal blood responsible for the morphofunctional status of hepatocytes (3,7,13,15,19,33,42,43,52).
Carbohydrate, proteins and amino acids absorbed in the intestine, in addition to contributing with energy and structural elements for cell maintenance and renewal (15), also act indirectly by stimulating the production of factors such as insulin itself (17,20,21,22).Thus, it is to be expected that prolonged nutritional deprivation and the consequent alterations in metabolism and hormonal homeostasis will affect liver trophism and the maintenance of liver structure by decreasing insulin availability (4,9,11,48), by changing the insulin/glucagon molar ratio (12,14,48,49), and by reducing portal speed and flow (31).In models of regenerative stimulation by partial hepatectomy, it has been demonstrated that malnutrition, and protein malnutrition 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 weight in the animals used in the present experiment (Table 3), similar to the 23.1 % value obtained by ADDIS et al. (1) and the 18% value reported by SKULLMAN et al. (41).It is interesting to note that kidney and spleen masses were decreased in proportions similar to body weight loss, but that the liver, an organ inserted in the portal circuit, presented a proportionally greater decrease, so that its relation to body weight was significantly modified (p<O.OOI) (Tables 2 and 3), a fact also observed by SKULLMAN et al. (41).
Work by ADDIS et al. (1,2) has shown that the liver, among all organs, loses greater amounts of protein at a faster rate, with a 20% loss within as little as 2 days.We believe that the "preferential" consumption of this protein reserve is also due to another additional effect.It is known that shunt of the hepatopetal flow is accompanied by Iiver atrophy (37,38), a fact attributed to the reduction of hepatotrophic factors such as insulin (33,43).Thus, the prolonged lack of food ingestion reducing the generation of hepatotrophic factors, creates a condition of relative insulin insufficiency (19), and possibly other factors, similar in its effects to that provoked by surgical shunt of hepatopetal flow.This fact is probably responsible for the greater reduction in liver mass in relation to body emaciation and mass of kidney and spleen, organs that do not depend directly on the splanchnic blood, in an additive Table 5 reports the percent variation in total DNA mass and protein mass per liver in the animals of group B between the beginning of the experiment (estimated values) and the end of the 7-day period of starvation.action with respect to mass reduction that would be expected as a function of simple protein consumption caused by malnutrition.The role of glucagon is less clear since reports that have related it to the trophic/regenerative stimulus are sometimes conflicting, now showing that glucagon is important for this process in rats and mice (3,7,18,27), now showing that it plays no part in dogs (3,44,45).However, several studies have shown that glucagon plays at least an adjuvant role in the generation of the stimulus by mechanisms such as synergism with insulin (7,51), maintaining glucose supply in the presence of high insulin levels (12), stimulating amino acid transport ( 16) or increasing portal flow (28,32), actions that are certainly altered by prolonged fasting.
The reduction in liver mass seems to have occurred in two ways, i.e., by a decrease in cell number (hypoplasia) reflected by the decrease in liver size with small variations in tissue DNA and protein concentrations (Table 4) or by the significant reduction in total DNA and proteins (Table 5), and by the reduction in size of the remaining hepatocytes (atropy), as suggested by the increased number of nuclei per area (p<O.OOI, Table 4), an indirect measurement of cell size, and by the significant decrease (p<O.OOI) of the proteinlDNA ratio.A decrease in this ratio and a similar conclusion were reported by YOUNG et al. (53) in a study of low-calorie diets.The decrease in hepatocyte size by reduced insulin availability has been previously shown by STARZL et al. (42,43) in experiments with selective portal infusions.
It is also interesting to note that the reduction in hepatic tissue protein per liver (-34.64%,Table 5) was significantly greater (p<O.OO1) than the reduction in DNA (-21.81%), a value that roughly represents the number of hepatocytes.Since this value is proportionally similar to the decrease in body weight (19.22%) and kidney weight (18.68%), it may be assumed that hypoplasia (decreased number of cells) was mainly due to prolonged malnutrition, accompanying the process of general consumption, whereas atrophy (reduction in cell size) was due to a reduction of the hepatotrophic stimulus, i.e., to the reduced availability of factors mainly represented by insulin.Although speculative, this hypothesis agrees with data reported by SIEGEL et al. (39,40) who were able to separate and individualize the hyperplastic and hypertrophic stimuli, the latter being more related to the availability of portal factors.In heterotopic transplants of autologous liver lobes associated with Eck fistulae, these investigators showed an attenuation of the cell atrophy process compared to controls without Eck fistulae, possibly because portal factors such as insulin, which is largely extracted during its first passage through the hepatic bed (5,30,31), by not being consumed as a function of the portocaval shunt, may reach the autografts via the arteries in a larger proportioR.
The process described here, i.e., hepatic hypoplasia and atrophy, due to malnutrition with the probable participation of a decreased supply of hepatotrophic factors, is in contrast to data observed in previous studies in which the intraperitoneal (portal) infusion of additional exogenous hepatotrophic factors caused an increase of as much as 86% in the hepatic mass of intact livers, with a mean value of 67.54% (35,36) reprodudng, in a way, the increase in liver mass observed when a liver is transplanted into a recipient of larger size than the donor (23,25,26,50).This shows that, from a morphological viewpoint, the liver and its hepatocytes are extremely sensible to the variations in trophic stimuli, and this fact may eventually be used in therapeutic manipulations of different Iiver diseases.
Investigations on the functional aspects of these situations are currently under way in our laboratory.

Table 3
reports the percent decrease in body and organ weight caused by malnutrition in group B animals.

Table 5 Total DNA and protein contents (mg/liver) before and after malnutrition in group B animals Table 3 Emaciation and decrease in organ weight in group
B animals (%)