Microquantitative determination of the distribution patterns of alcohol dehydrogenase activity in the liver of rat, guinea-pig and horse

Microquantitative measurements of ADH-activity were carried out on the livers of male and female rats, guinea-pigs and horses (two geldings and a mare). Lyophilized cryostat sections of liver parenchyma were microdissected the whole way along the sinusoidal length from the terminal afferent vessels to the terminal efferent venule. ADH activity in samples of about 50-150 ng was measured in a microbiochemical assay using the oil-well technique without enzymatic cycling, by direct luminometric determination of NADH. On the basis of the single measurements, mean values of total hepatic ADH activity could be calculated and the specific distribution patterns graphically demonstrated. Total activity of ADH in the liver of the female rat is 1.6 times higher than in the male; the male distribution pattern exhibits a relative maximum in the intermediary zone of the acinus while the activity in the liver of female rats increases towards a perivenous maximum. Mean values for total ADH activity in the livers of male and female guinea-pigs are almost equal and there is, moreover, no clear intra-acinar gradient. Mare and castrated male horses show high hepatic ADH activity which is evenly distributed in the liver acinus.     

Microquantitative determination of intra-acinar distribution profiles of low-Km and high-Km aldehyde dehydrogenase activity in rat liver

Microquantitative measurements of total and of low-Km aldehyde dehydrogenase (ALDH) activity with millimolar and micromolar concentrations of acetaldehyde and propionaldehyde were carried out on the livers of male and female rats. Lyophilized cryostat sections of liver parenchyma were microdissected along the entire sinusoidal length from the terminal afferent vessels to the terminal efferent venule. ALDH activity was measured in a microbiochemical assay using the oil-well technique with luminometric determination of NADH. On the basis of single measurements, mean values of total, low-Km and high-Km ALDH activity could be calculated and the specific distribution patterns graphically demonstrated. The two substrates acetaldehyde and propionaldehyde yielded similar values of ALDH activity, the intraacinar distribution profiles of which showed characteristic sex differences. In the liver of the male rat high-Km ALDH activity has two flat peaks in the periportal and the perivenous area, while low-Km ALDH activity is almost evenly distributed throughout the acinus. In the livers of female rats, both high-Km and low-Km ALDH activity shows a continuous gradient which decreases from the periportal to the perivenous zone (pp/pv = 1.4:1). It was therefore possible to demonstrate that the maxima of alcohol dehydrogenase activity and of low-Km ALDH activity are localized in opposite parts of the liver acinus of the female rat. This heterotopy should have consequences with respect to hepatotoxicity after alcohol ingestion.     

The effects of starving and refeeding on the intra-acinar distribution pattern of alcohol-dehydrogenase activity in rat liver

Microquantitative determinations of ADH activity were carried out on the livers of male and female rats. The animals were either starved for 84 h, or starved and then refed with a carbohydrate-rich diet for 6 nights. When the enzyme activity is expressed in mumoles/min/g dry weight, fasting does not appear to alter liver ADH activity, while in starved and subsequently refed rats it is diminished by 20%. Microquantitative measurements of ADH activity in 50-150 ng lyophilized tissue samples, microdissected the whole way along the sinusoidal length, made the computer-aided plotting of intra-acinar distribution patterns possible. The results showed that, under the feeding conditions selected, only minor changes in the ADH activity profiles occur in the liver acinus. These are within the range of the standard deviations of the normal mean values. From these results it can be deduced that fasting and refeeding do not lead to specific inhibition or induction of liver ADH activity. - The decrease of ADH activity of total liver (mumol/min) per total body weight in starved rats is obviously the result of a loss of protein which affects the liver cells of all acinar zones almost equally.     

The effects of starving and refeeding on the intra-acinar distribution pattern of alcohol-dehydrogenase activity in rat liver

Summary Microquantitative determinations of ADH activity were carried out on the livers of male and female rats. The animals were either starved for 84 h, or starved and then refed with a carbohydrate-rich diet for 6 nights. When the enzyme activity is expressed in moles/min/g dry weight, fasting does not appear to alter liver ADH activity, while in starved and subsequently refed rats it is diminished by 20%. Microquantitative measurements of ADH activity in 50–150 ng lyophilized tissue samples, microdissected the whole way along the sinusoidal length, made the computeraided plotting of intra-acinar distribution patterns possible. The results showed that, under the feeding conditions selected, only minor changes in the ADH activity profiles occur in the liver acinus. These are within the range of the standard deviations of the normal mean values. From these results it can be deduced that fasting and refeeding do not lead to specific inhibition or induction of liver ADH activity.—The decrease of ADH activity of total liver (mol/min) per total body weight in starved rats is obviously the result of a loss of protein which affects the liver cells of all acinar zones almost equally.This study is part of a dissertation which will be presented to the Phil.-Nat. Faculty of the University of Basel by I.P. MalySupported by grants from the Schweizerische Stiftung für Alkoholforschung     

Sexual dimorphic expression of ADH in rat liver: importance of the hypothalamic-pituitary-liver axis

Hepatic alcohol dehydrogenase (ADH) activity is higher in female than in male rats. Although sex steroids, thyroid, and growth hormone (GH) have been shown to regulate hepatic ADH, the mechanism(s) for sexual dimorphic expression is unclear. We tested the possibility that the GH secretory pattern determined differential expression of ADH. Gonadectomized and hypophysectomized male and female rats were examined. Hepatic ADH activity was 2.1-fold greater in females. Because protein and mRNA content were also 1.7- and 2.4-fold greater, results indicated that activity differences were due to pretranslational mechanisms. Estradiol increased ADH selectively in males, and testosterone selectively decreased activity and mRNA levels in females. Effect of sex steroids on ADH was lost after hypophysectomy; infusion of GH in males increased ADH to basal female levels, supporting a role of the pituitary-liver axis. However, GH and L-thyroxine (T4) replacements alone in hypophysectomized rats did not restore dimorphic differences for either ADH activity or mRNA levels. On the other hand, T4 in combination with intermittent administration of GH reduced ADH activity and mRNA to basal male values, whereas T4 plus GH infusion replicated female levels. These results indicate that the intermittent male pattern of GH secretion combined with T4 is the principal determinant of low ADH activity in male liver.     

Phosphoenolpyruvate carboxykinase activity patterns in the liver acinus of diabetic and diabetic and estrogen treated rats

Summary The acinar activity pattern of phosphoenolpyruvate carboxykinase (PEPCK) was investigated in livers of streptozotocin diabetic male and female rats and in addition in livers of diabetic males, which had undergone estrogen treatment. In all diabetic animals blood glucose levels were supranormal and liver PEPCK activity was increased. This increase in activity was greatest in estrogen treated diabetic males and lowest in diabetic females. Plasma insulin levels were reduced after the application of streptozotocin to otherwise normal male and female rats. Yet, in males treated in addition with estrogens the plasma insulin levels reached the normal range again. The PEPCK activity showed a heterotopic distribution along the acinus. The periportal to perivenous gradient was steeper in males compared to females in the untreated as well as in the diabetic state. The application of estrogens to males resulted in a further steepening of the gradient.     

The intra-acinar distribution patterns of alcohol-dehydrogenase activity in the liver of juvenile, castrated and testosterone-treated rats

Rat liver alcohol dehydrogenase shows characteristic sex-differences with respect to activity and heterotopy. For the recognition of gonadal influences on the intra-acinar distribution patterns luminometric determinations of ADH activity were carried out on 50-150 ng lyophilized liver tissue samples which had been microdissected along the sinusoidal length. Juvenile rats of both sexes showed equally high alcohol dehydrogenase activity, which surpassed the adult values by a factor of 2 in males and 1.3 in females. The distribution pattern was rather flat, with a weak maximum at the beginning of the last third of the sinusoid. Castration of adult male and female rats resulted in an increase of alcohol dehydrogenase activity to around the prepubertal values. The intra-acinar profiles showed a gradual increase in activity from low periportal values to a peak near the perivenous zone. Only the hepatocytes directly adjacent to the efferent venule showed an even lower activity. Administration of testosterone to castrated animals had no effect on the ADH activity in males and resulted in only a slight decrease of enzyme activity in females. The intra-acinar distribution patterns showed an intermediary peak at the end of the second third of the sinusoidal length in males and a gradual increase of activity, beginning periportally, in the direction of the perivenous zone in females. The present findings on total activity of ADH and its distribution patterns in the liver are considered to be the result of complex hormonal alterations rather than a specific effect of testosterone.     

Phosphoenolpyruvate carboxykinase activity patterns in the liver acinus of diabetic and diabetic and estrogen treated rats

The acinar activity pattern of phosphoenolpyruvate carboxykinase (PEPCK) was investigated in livers of streptozotocin diabetic male and female rats and in addition in livers of diabetic males, which had undergone estrogen treatment. In all diabetic animals blood glucose levels were supranormal and liver PEPCK activity was increased. This increase in activity was greatest in estrogen treated diabetic males and lowest in diabetic females. Plasma insulin levels were reduced after the application of streptozotocin to otherwise normal male and female rats. Yet, in males treated in addition with estrogens the plasma insulin levels reached the normal range again. The PEPCK activity showed a heterotopic distribution along the acinus. The periportal to perivenous gradient was steeper in males compared to females in the untreated as well as in the diabetic state. The application of estrogens to males resulted in a further steepening of the gradient.     

NADP-dependent dehydrogenases in rat liver parenchyma. II. Comparison of qualitative and quantitative G6PDH distribution patterns with particular reference to sex differences.

Qualitative histochemical G6PDH distribution patterns obtained in the liver acinus of adult male and female rats with an improved method (Rieder et al., 1978) served as a basis for the isolation by microdissection of tissue samples of defined zonal affiliation. G6PDH activity was assayed quantitatively in tissue samples of zones 1 and 3 by a microfluorometric method, using the oil well technique and enzymatic cycling (Burch et al., 1963; Lowry and Passonneau, 1972). With the use of a correlation system further evidence could be presented for the validity of the recently described qualitative distribution patterns. From a total of 50 analyzed tissue samples the following G6PDH activities were calculated: 4.25 +/- 1.56 U/g dry weight in zone 1 and 2.08 +/- 0.46 U/g dry weight in zone 3 of male and 7.21 +/- 1.03 U/g dry weight in zone 1 and 11.10 +/- 2.56 U/g dry weight in zone 3 of female rats. These data were corrected for interference from the G6PDH activity of the Kupffer cells within zone 1 samples (approximately 80 U/g dry weight), so that the actual relative values for the parenchymal activity could be estimated for the first time: 2 U/g dry weight in zones 1 and 3 of male animals, 5 U/g dry weight in zone 1 and 11 U/g dry weight in zone 3 of female animals. In female livers G6PDH activity in zone 1 is therefore 2.5 times higher, and in zone 3 5 times higher than in the male. These zonal as well as sex-differences are clearly indicative of a heterogeneous functional organization of the liver acinus in terms of capacity for NADPH production, mainly in connection with reductive reactions in fatty acid synthesis.     

Sex differences of the influence of T3 on the topical distribution of phosphoenolpyruvate carboxykinase activity in the liver acinus

Summary Phosphoenolpyruvate carboxykinase (PEPCK) activity was investigated in livers of triiodothyronine (T₃) treated male and female rats with special regard to its intraacinar localization. In untreated controls of both, male and female rats, the activity was heterotopically distributed within the acinus with highest values in the periportal zone, and with lowest values in the perivenous zone. This periportal to perivenous activity gradient revealed to be under the influence of T₃. Application of T₃ resulted in a relative increase of PEPCK activity which was much greater in the livers of females than in males. The extent of T₃-induced augmentation of PEPCK activity was dependent on the intraacinar position. In both sexes greatest relative activation was found in the perivenous zone. In female animals, the perivenous activity of T₃ treated livers was comparable to that observed in the periportal zone of controls.     

NADP-dependent dehydrogenases in rat liver parenchyma

Summary Qualitative histochemical G6PDH distribution patterns obtained in the liver acinus of adult male and female rats with an improved method (Rieder et al., 1978) served as a basis for the isolation by microdissection of tissue samples of defined zonal affiliation. G6PDH activity was assayed quantitatively in tissue samples of zones 1 and 3 by a microfluorometric method, using the oil well technique and enzymatic cycling (Burch et al., 1963; Lowry and Passonneau, 1972). With the use of a correlation system further evidence could be presented for the validity of the recently described qualitative distribution patterns. From a total of 50 analyzed tissue samples the following G6PDH activities were calculated: 4.25±1.56 U/g dry weight in zone 1 and 2.08±0.46 U/g dry weight in zone 3 of male and 7.21±1.03 U/g dry weight in zone 1 and 11.10±2.56 U/g dry weight in zone 3 of female rats. These data were corrected for interference from the G6PDH activity of the Kupffer cells within zone 1 samples (approximately 80 U/g dry weight), so that the actual relative values for the parenchymal activity could be estimated for the first time: 2 U/g dry weight in zones 1 and 3 of male animals, 5 U/g dry weight in zone 1 and 11 U/g dry weight in zone 3 of female animals. In female livers G6PDH activity in zone 1 is therefore 2.5 times higher, and in zone 3 5 times higher than in the male. These zonal as well as sex-differences are clearly indicative of a heterogeneous functional organization of the liver acinus in terms of capacity for NADPH production, mainly in connection with reductive reactions in fatty acid synthesis.Supported by a grant from the SFB 46 (Molgrudent)     

Microbiochemical approach to liver cell heterogeneity around terminal hepatic venules

Using lyophilized cryostat sections of liver the activities of alanine aminotransferase, lactate dehydrogenase, and pyruvate kinase were measured using a Lowry technique in the first layer of hepatocytes adjacent to terminal hepatic venules and in the residual parenchymal of the perivenous zone of the acinus in normally fed adult male Wistar rats. Alanine aminotransferase was homogeneously distributed in the two areas measured (ratio hepatocytes adjacent to terminal hepatic venules/residual parenchyma of the perivenous zone: 1.05). Enzyme activities of the lactate dehydrogenase were significantly lower in the hepatocytes adjacent to terminal hepatic venules (ratio: 0.65) and those of the pyruvate kinase significantly higher (ratio: 1.12) than in the residual parenchyma of the perivenous zone indicating liver cell heterogeneity in this zone of the liver acinus.     

A comparison by species, age and sex of cysteinesulfinate decarboxylase activity and taurine concentration in liver and brain of animals

Cysteinesulfinate decarboxylase activity and taurine concentration were determined in liver and brain of rats, mice, cats, guinea-pigs and sheep. Values were compared for male and female animals and in some cases measurements were also made in animals of different ages. Cysteinesulfinate decarboxylase activity and taurine concentration were also measured in liver and brain of male and female rat pups during the postnatal period. Hepatic cysteinesulfinate decarboxylase activity increased in both male and female rat pups during the postnatal period and then declined markedly in female rats so that activity in adult males was 16-fold that in adult females. Cysteinesulfinate decarboxylase activity in liver of 5- to 6-week old kittens was 73 times that observed in liver of 15-month old cats. Taurine level in liver of guinea-pigs was much lower than that in liver of any other species studied.     

The effects of acetylsalicylic acid on phosphoenolpyruvate carboxykinase activity and acinar heterotopy in livers from juvenile and adult rats

Summary Male and female juvenile as well as adult rats were treated with acetylsalicilic acid in order to examine the effect of the drug on over-all activity and activity distribution of phosphoenolpyruvate carboxykinase (PEPCK) in the liver acinus. Upon administration of acetylsalicilic acid PEPCK activity increased in juvenile males and adult females, but was reduced in juvenile females and adult males. The periportal-perivenous activity gradient along the sinusoidal length, which is flatter in untreated juvenile rats compared to the livers of adult rats, was distinctly steepened by acetylsalicilic acid treatment. Acetylsalicilic acid did not affect the gradient in adult rats.     

The effects of acetylsalicylic acid on phosphoenolpyruvate carboxykinase activity and acinar heterotopy in livers from juvenile and adult rats

Male and female juvenile as well as adult rats were treated with acetylsalicylic acid in order to examine the effect of the drug on over-all activity and activity distribution of phosphoenolpyruvate carboxykinase (PEPCK) in the liver acinus. Upon administration of acetylsalicylic acid PEPCK activity increased in juvenile males and adult females, but was reduced in juvenile females and adult males. The periportal-perivenous activity gradient along the sinusoidal length, which is flatter in untreated juvenile rats compared to the livers of adult rats, was distinctly steepened by acetylsalicylic acid treatment. Acetylsalicylic acid did not affect the gradient in adult rats.     

Microbiochemical investiagation on diurnal rhythmic changes of the activities of the lactate dehydrogenase in the periportal and perivenous zones of the acinus of the rat liver

Summary Activities of the lactate dehydrogenase within the periportal zone and within the perivenous zone in the first layer of hepatocytes adjacent to terminal hepatic venules and the remainder of the perivenous parenchyma of the liver acinus were measured using a Lowry technique during a full 24-h cycle (08.00-08.00) in untreated adult male Wistar rats kept under 12 h of light and 12 h of darkness, scotophase 18.25-06.25. In all three regions studied a broad first maximum was recorded between 10.00 and 22.00 with the peak value at 16.00 and a high and narrow peak at 24.00. Zonal and intrazonal heterogeneity of the lactate dehydrogenase were retained during the full day and night cycle. The regions displayed individual dynamic changes in enzyme activity.Supported by the Deutsche Forschungsgemeinschaft (Hi 318/21)     

The circadian rhythm of intra-acinar profiles of alcohol dehydrogenase activity in rat liver: a microquantitative study

Summary Using microquantitative measurements of alcohol dehydrogenase activity in microdissected samples of liver tissue along the sinusoidal length, the intra-acinar distribution profiles were studied in seven groups of female rats at different times during 24h with a light phase from 630h to 1830h. The mean values of alcohol dehydrogenase activity showed a circadian rhythm with a minimum at 13.30h and a maximum at 17.30h (p<0.0001). However, the intra-acinar gradients remained almost unchanged, indicating that increase and decrease in enzyme activity takes place simultaneously in all parts of the liver acinus. This observation, together with data from the literature, suggests that the circadian rhythm of alcohol dehydrogenase activity reflects variations in different liver cell consituents, rather than enzyme protein synthesis or proteolysis.     

Effects of high-dose ethinyl estradiol on serum concentrations and hepatic secretion of the very-low-density lipoprotein, triacylglycerol, cholesterol, and apolipoprotein A-I in the rat

Female and male rats were treated with ethinyl estradiol (5.0 mg/kg daily for 5 days). Control animals were pair fed to compensate for the reduction in food intake induced by the estrogen, or were fed ad libitum. Treatment with ethinyl estradiol reduced total cholesterol and apolipoprotein A-I concentrations in the serum of female and male animals. The concentrations of serum and hepatic triacylglycerol were depressed markedly in animals of both sexes in groups treated with ethinyl estradiol, compared to the control group fed ad libitum. Compared to the pair-fed controls, however, ethinyl estradiol had only a very minor further reduction on serum triacylglycerol concentration. In male and female rats, the synthesis and secretion of triacylglycerol by the liver was, in comparison to the pair-fed controls, stimulated by estrogen, whereas the secretion of unesterified cholesterol was unaffected by any of the treatment regimens. The synthesis and secretion of total cholesteryl esters by livers from male and female rats was increased by treatment with ethinyl estradiol. The hepatic synthesis and secretion of VLDL triacylglycerol and cholesteryl ester was stimulated by ethinyl estradiol in male and female rats, and the VLDL particle was enriched with cholesteryl ester. Treatment with the high-dose estrogen increased the secretion of apolipoprotein A-I by livers from female rats. It is suggested that the depression in the serum concentrations of cholesteryl esters and apolipoprotein A-I is the result of increased rates of hepatic and/or peripheral catabolism of these components and that the hepatic production rates were increased or unaffected in animals administered high doses of ethinyl estradiol. Since the secretion of apolipoprotein A-I by livers from male rats was unaffected by treatment with ethinyl estradiol, the response to estrogen may be sex related.     

Changes in hepatic lipogenesis during development of the rat

1. Changes in the activities of ATP citrate lyase, ;malic' enzyme, glucose 6-phosphate dehydrogenase, pyruvate kinase and fructose 1,6-diphosphatase, and in the ability to incorporate [1-(14)C]acetate into lipid have been measured in the livers of developing rats between late foetal life and maturity. 2. In male rats the activities of those systems directly or indirectly concerned in lipogenesis (acetate incorporation into lipid, ATP citrate lyase and glucose 6-phosphate dehydrogenase) fall after birth and are maintained at a low value until weaning. After weaning these activities rise to a maximum between 30 and 40 days and then decline, reaching adult values at about 60 days. ;Malic' enzyme activity follows a similar course, except that none could be detected in the foetal liver. Pyruvate kinase activity is lower in foetal than in adult livers and rises to slightly higher than the adult value in the post-weaning period. Fructose 1,6-diphosphatase activity rises from a very low foetal value to reach a maximum at about 10 days but falls rapidly after weaning to reach adult values at about 30 days. 3. Weaning rats on to a high-fat diet caused the low activities of acetate incorporation, ATP citrate lyase, glucose 6-phosphate dehydrogenase and pyruvate kinase, characteristic of the suckling period, to persist. ;Malic' enzyme and fructose 1,6-diphosphatase activities were not altered appreciably. 4. No differences could be detected in hepatic enzyme activities between males and females up to 35 days, but after this time female rats gave higher values for acetate incorporation, glucose 6-phosphate dehydrogenase activity and ;malic' enzyme activity. 5. The results are discussed in relation to changes in alimentation and hormonal influences.     

Distribution of cyclic AMP phosphodiesterase in microdissected periportal and perivenous rat liver tissue with different dietary states.

Cyclic AMP phosphodiesterase was measured in liver homogenates and microdissected periportal and perivenous liver tissue from rats in different dietary states under different conditions of substrate saturation and effector stimulation. A radiochemical microtest, more sensitive by 2-3 orders of magnitude than the usual assay, was established for the determination of the activity in liver samples corresponding to 200-800 ng dry weight. At saturating cyclic AMP concentrations (46 microM) phosphodiesterase was homogeneously distributed within the liver acinus of fed rats. Starvation for 48 h led to a decrease in the overall activity and to a heterogenous distribution with slightly higher activities in the perivenous zone. At physiological cyclic AMP concentrations (1.8 microM) phosphodiesterase showed a flat zonal gradient in livers of fed rats with higher levels in the periportal zone; after 48 h starvation it was homogeneously distributed. In the presence of cyclic GMP (2 microM) the basal activity at physiological substrate concentrations was stimulated to a greater extent in the perivenous zone. This led to a homogeneous activity distribution in the fed state and to a heterogenous pattern with a slight perivenous maximum in the fasted state. Thus there was no or only a small zonal heterogeneity of signal transmitting enzymes such as cyclic AMP phosphodiesterase and glucagon-stimulated adenylate cyclase (Zierz and Jungermann 1984). This similar signal transducing capacity in the periportal and the perivenous area will contribute to maintain the zonation of signal input due to the hormone concentration gradients across the liver acinus.