Occurrence of Lipids in the Liver of the Hypertriglyceridemic Rats

Aims: The purpose of this study was to demonstrate the accumulation and distribution of lipids in the liver of the adult Prague hereditary hypertriglyceridemic (HHTg) rats. They reveal an increased expression of 11β-hydroxysteroid dehydrogenase 1 (11HSD1), which locally increases concentration of corticosterone in the liver. We studied the effect of the 11HSD1 inhibition on the lipid content. Methods: Samples of liver of three groups of adult female rats – HHTg, HHTg treated for 14 days with 50 mg/kg/day carbenoxolone (HHTg+CBX) and control Wistar rats, were examined histochemically. Cryosections of the samples were stained with Oil red O or Sudan black B to demonstrate different kinds of lipids. Extent and intensity of staining was evaluated semiquantitatively. Results: The orientational analysis showed a higher extent and intensity of the staining of the liver of HHTg and HHTg+CBX rats (equal in both hypertriglyceridemic groups) than that of the control Wistar rats. Oil red O stained unsaturated fatty acids and neutral fats, mainly triglycerides. The difference was on average 30 per cent. Staining of phospholipids with Sudan black B showed similarly the higher positivity in the hypertriglyceridemic groups than in controls. Conclusions: Staining for triglycerides and phospholipids demonstrated a higher amount of lipids in the liver of HHTg and HHTg+CBX female rats than in controls. The inhibition of 11HSD1 activity had no effect on the lipid content in the liver of the HHTg rats.


INTRODUCTION
The increasing number of people in advanced countries suffers from a cluster of associated metabolic disorders that are collectively termed metabolic syndrome and that include hypertension, glucose intolerance, hyperinsulinemia, hypertriglyceridemia, hepatic steatosis and obesity.Similar symptoms can be found in patients with Cushing's syndrome which is characterised by elevated plasma level of glucocorticoids and thus the disturbances of metabolic syndrome seem to be not only insulin -but also steroid-sensitive.
11β-hydroxysteroid dehydrogenase 1 (11HSD1) is an oxidoreductase highly expressed in liver and adipose tissue catalysing an interconversion of corticosterone into 11-dehydrocorticosterone and vice versa.Both activities, reductase and oxidase of 11HSD1, were found in vitro, however conversion of 11-dehydrocorticosterone to corticosterone predominates in intact tissues 1 .Study on transgenic mice with selective overexpression of 11HSD1 in liver, a non-obese model of metabolic syndrome, showed enormous hepatic steatosis in these animals 2 .
Prague hereditary hypertriglyceridemic rats (HHTg) were previously described as a genetic model of metabolic syndrome.They suffer from hypertriglyceridemia, insulin resistance and hypertension 3 .Our pilot experiments revealed an increased activity and expression of 11HSD1 in the liver of female HHTg rats.We therefore decided to study the role of 11HSD1 in accumulation and distribution of lipids in the liver of these animals using chronic administration of carbenoxolone (CBX), a nonselective inhibitor of steroid dehydrogenases.We examined the lipid content in the rat liver using lipid histochemistry.

Experimental groups
Lipid content was investigated in the liver of three experimental groups with 10 animals: adult Prague hereditary hypertriglyceridemic rats (HHTg), HHTg rats treated with carbenoxolone (14 days, 50 mg/kg/day dissolved in drinking water, HHTg +CBX), and Wistar rats as normotriglyceridemic controls.

Histochemistry of lipids in the liver
Animals were killed by decapitation, liver were removed, immediately frozen in liquid nitrogen and stored until use at -84 °C.They were processed for cryosections 8 μm thick, fixed in Baker solution for 1 hour, rinsed in water (tap water followed by distilled water), rinsed in 50 per cent ethanol and stained -either with Oil red O or Sudan black B 4  Explanations: HHTg -Prague hereditary hypertriglyceridemic rats; HHTg + CBX -HHTg rats treated with carbenoxolone; extent and intensity of staining: strong "+", mild "±", weak, or none "(±)-0"; n -number of animals processed for histological examination.The dyes stained different kinds of lipids: Oil red stained unsaturated fatty acids and neutral lipids (mostly triglycerides).Sudan black coloured predominantly phospholipids in fixed sections.Evaluation of the colouring extent and intensity was performed semiquantitatively, discriminating strong "+", mild "±", and weak, hardly visible or none colouring as "(±)" or "0" (Table 1).The strong colouring exceeded more than half of the section area, the mild colouring occupied medium extent and intensity of the section and the weak colouring was that, which was rare and hardly visible.The staining procedure was repeated in several sections of any sample to get representative results.Besides extent and intensity of the section staining we still evaluated its distribution in the liver sections and localization of lipid droplets in various cell types.

Fig. 1 A
Fig. 1 A. Oil red O staining of lipid droplets in hepatocytes (full arrows) and Ito cells (arrowheads) of the liver of HHTg rat; bar = 100 μm.

Fig. 1 B
Fig. 1 B. Sudan black B dark blue staining of phospholipids in perisinusoidal localization in the periportal zone of the liver lobule in HHTg rat (arrows); bar represents 100 μm.

Table 1 .
. Staining with Oil red O (Sigma) saturated in 70 per cent ethanol lasted 1 hour.The sec-Z.Zemanova, M. Strnadova, Z. Jirsova, P. Klusonova Intensity and area of the lipid staining in sections of the liver samples (percentage of the total).