Matti T. Kähönen

Hypolipidemia and peroxisome proliferation induced by phenoxyacetic acid herbicides in rats

Male Wistar rats were treated daily by gavage with two phenoxy herbicides, 2,4-dichlorophenoxyacetic acid (2,4-D)(100-200 mg/kg body wt) and 4-chloro-2-methylphenoxyacetic acid (MCPA) (100-200 mg/kg body wt), and with the chemically different glyphosate N-phosphonomethyl glycine (300 mg/kg body wt) 5 days per week for 2 weeks. A hypolipidemic drug, clofibrate [ethyl-2-(4-chlorophenoxy)-2-methylpropionate], which is structurally related to phenoxy acids, was used as a positive control (200 mg/kg body wt). 2,4-D and MCPA had several effects similar to those of clofibrate: all three compounds induced proliferation of hepatic peroxisomes, decreased serum lipid levels, and increased hepatic carnitine acetyltransferase and catalase activities. 2,4-D and MCPA, but not clofibrate, decreased lipoprotein lipase activity in the adipose tissue to about a third of the control value but did not change the lipoprotein lipase activity in the heart muscle. The data suggest that these compounds cause hypolipidemia not by enhancing the storage of peripheral lipids in adipose tissue but by preferentially increasing lipid utilization in the liver. Glyphosate caused no peroxisome proliferation or hypolipidemia, suggesting that these effects are associated with the structural similarity between phenoxy acid herbicides and clofibrate.

Effect of clofibrate treatment on carnitine acyltransferases in different subcellular fractions of rat liver

The subcellular distribution of carnitine acetyl-, octanoyl-, and palmitoyl- transferase in the livers of normal and clofibrate-treated male rats was studied with isopycnic sucrose density gradient fractionation. In normal liver 48% of total carnitine acetyltransferase activity was peroxisomal, 36% of the activity located in mitochondria and 16% in a membranous fraction containing microsomes. Carnitine octanoyltransferase and carnitine palmitoyltransferase were confined almost totally (77--81%) to mitochondria in normal liver. Clofibrate treatment increased the total activity of carnitine acetyltransferase over 30 times, whereas the total activities of the other two transferases were increased only 5-fold. From the three different subcellular carnitine acetyltransferases the mitochondrial one was most responsive to clofibrate treatment, i.e. the rise in mitochondrial activity was over 70-fold as contrasted to the 6- and 14-fold rises in peroxisomal and microsomal activities, respectively. After treatment mitochondria contained 79% of total activity. It is concluded that the clofibrate-induced increase of carnitine acetyltransferase activity is not due to the peroxisomal proliferation that occurs during clofibrate treatment. The rise in peroxisomal activity contributed only 8% to the total increase. After clofibrate treatment the greatest part of carnitine octanoyl- and palmitoyltransferase activities were located in mitochondria but a considerable amount of both activities was found also in the soluble fraction of liver.

Effect of clofibrate and gemfibrozil on the activities of mitochondrial carnitine acyltransferases in rat liver dose-response relations

The effects of different doses of clofibrate and gemfibrozil on liver size, serum triglyceride concentration and the activities of hepatic mitochondrial alpha-glycerophosphate dehydrogenase (alpha-GPD) and carnitine acyltransferases were studied in male rats. Both clofibrate and gemfibrozil treatment effectively decreased the fructose-induced hypertriglyceridaemia and increased the liver to body weight ratio. Clofibrate treatment also induced an increase of many times in the activities of mitochondrial alpha-GPD and carnitine acyltransferases, the effect increasing with the dose used. The effect of gemfibrozil on the activities of the enzymes was significantly smaller. There was no correlation between the decrease in serum triglyceride concentration and the changes in the activities of the enzymes. Only clofibrate increased the rate of fatty acylcarnitine oxidation in isolated mitochondria. It is concluded that both drugs increased the size of the rat liver, but that only clofibrate influenced the mitochondrial enzyme activities of mitochondrial carnitine acyltransferases and the accelerated mitochondrial oxidation of fatty acids are not the mechanisms by which these drugs lower serum lipid levels.

Ethanol metabolism in rats treated with ethyl-α-p-chlorophenoxyiso-butyrate (Clofibrate)

Clofibrate treatment (60 mg/day/100 g body wt im. for nine days) increased the liver to body weight ratio of the rats by 71 %. The activity of mitochondrial α-glycerophosphate dehydrogenase in the liver of clofibrate-treated rats was five times greater than in normal rat liver. The amounts of cytochrome a and mitochondrial protein per gram of liver wet weight were not changed. Clofibrate treatment increased the oxygen consumption of liver slices by 16 %. Ethanol increased the lactate/pyruvate ratio (an index of the redox state in the cytosolic compartment of the liver cell) in perfused normal livers and only slightly in the livers from clofibrate-treated rats. This effect of clofibrate is similar to that of thyroxine and seems to be due to enzyme induction, as diethylaminoclofibrate hydrochloride added in vitro did not prevent ethanol-induced increase in hepatic lactate/pyruvate ratio. Clofibrate treatment in rats significantly increased the disappearance rate of ethanol. The activity of hepatic alcohol dehydrogenase per gram of liver wet weight of per milligram of soluble protein was not changed by clofibrate treatment.

Regulation of cellular respiration by thyroid hormone. Spectroscopic evidence of mitochondrial control in intact rat liver

Abstract In order to elucidate the role of the mitochondrial control (coupling) mechanism in the regulation of respiration in intact liver, we investigated perfused rat livers using direct spectrophotometry. The acute effects of fructose on hepatic concentrations of adenine nucleotides, inorganic phosphate, and citrate were also studied. In hypothyroid rat livers fructose caused a small stimulation of respiration, followed by marked inhibition lasting about 10 min. During the respiratory inhibition the redox state of the flavoproteins was reduced but that of cytochrome b was markedly oxidized. In euthyroid rats fructose first stimulated hepatic oxygen consumption, but the subsequent inhibition of respiration was much smaller than that in hypothyroid rats. During the inhibitory phase flavoproteins were reduced, but the oxidation of cytochrome b was significantly less than in hypothyroid rats. In hyperthyroid rat liver, fructose stimulated respiration without any subsequent inhibition. Flavoproteins and all cytochromes were reduced, and no transient oxidation was observed in contrast to hypo- and euthyroid rats. In all groups of rats fructose induced a prompt decrease in hepatic concentrations of ATP and inorganic phosphate. Fructose did not induce any acute changes in the concentrations of citrate, but in hyperthyroid rat liver the citrate concentration was three times greater than in hypo- or euthryoid liver. These results suggest that, in intact tissue also, mitochondrial control is important in the regulation of energy metabolism during the metabolism of fructose. Thyroxine treatment seems to loosen this coupling, perhaps by increasing the concentration and oxidation of fatty acids in the liver.

Effect of clofibrate treatment on the activity of carnitine acetyltransferase in rat tissues

Carnitine acetyltransferase (CAT) (EC 2.3.1.7) is an enzyme catalyzing the reversible transfer of an acetyl group from acetyl-CoA to camitine. This enzyme is primarily located in the membranous part of the mitochondria [ 1 ] . The activity of the enzyme has been found to be high in heart, testis, brown adipose tissue and skeletal muscle but low in liver [2]. Fasting increases the activity of the liver enzyme [3]. In spite of this substantial ibformation about the behaviour and location of CAT, its physiological function is uncertain. Some investigators have proposed a role as an acetyl carrier throughout the cell [4] but this has been disputed by many authors who have suggested an acetyl group buffering function for CAT [5] . In either case, through acetyl group and camitine metabolism, it could influence both lipid and carbohydrate metabolism [6]. Clofibrate (ethyl-&p-chlorophenoxyisobutyrate) is a well-known drug decreasing the concentrations of plasma lipids, especially that of triglycerides [7]. It affects the hepatic metabolism of free fatty acids and triglycerides [8] and changes the activity of several enzymes [9,10]. Because CAT may have a central role in lipid metabolism, we have now studied the effect of clofibrate treatment on CAT activity in some rat tissues. Two different doses of clofibrate were administered, and the resulting changes in CAT activity were compared with the effect of thyroxine.

Metabolic interactions of fructose and ethanol in perfused liver of normal and thyroxine-treated rats.

Abstract Combined effects of thyroxine treatment and fructose on the rate of ethanol oxidation, the hepatic redox state and the hepatic oxygen consumption were studied in perfused rat liver. Neither thyroxine treatment nor fructose was found to influence the rate of ethanol elimination by the liver. An ethanol-induced increase in lactate/pyruvate concentration ratio in the perfusion medium was augmented by the simultaneous addition of fructose. Thyroxine treatment markedly inhibited this redox change. In perfused livers of thyroxine-treated rats, fructose and glucose were utilized significantly more rapidly than in normal livers. In both kinds of liver, fructose was metabolized about three times as fast as glucose. Lactate and pyruvate production from fructose was ten times that of glucose. Ethanol had no effect on the rate of fructose or glucose elimination in either group of rats. The glucose production from fructose was greater in normal rat livers than in thyroxine-treated ones and was inhibited by ethanol in normal livers. In perfused livers of normal rats, fructose had a biphasic effect on the respiration. During the first 2 min after the addition of fructose, a stimulation of respiration occurred. The oxygen consumption was then inhibited for 8 to 10 min. In normal rats, after an injection of fructose, the hepatic adenosine triphosphate (ATP) and inorganic phosphate (P i ) concentrations diminished coincidently with the respiratory inhibition observed in the perfusions. In perfused livers of hyperthyroid rats, fructose only stimulated the oxygen consumption, but in contrast to normal livers, no inhibition was observed. Hepatic ATP and P i concentrations in these rats were decreased by fructose but not as much as in normal livers. The fructose-induced changes in hepatic oxygen consumption and ATP and P i content are interpreted as an example of mutual control between glycolysis and oxidative metabolism. The mechanism of this phenomenon and its control by thyroid hormones are discussed.

Studies on the mechanism on inhibition of acute alcoholic fatty liver by clofibrate

Abstract The effect of ethanol on hepatic α-glycerophosphate (α-GP), dihydroxyacetone phosphate (DAP) and triglyceride (TG) concentrations, and on serum levels of triglycerides (TG) and free fatty acids (FFA) was studied 4 and 12 hr after administration of ethanol in normal and ethyl-α-p-chlorophenoxyisobutyrate (clofibrate)-treated rats. In the normal rats, ethanol increased liver α-GP and TG concentrations, α- GP DAP ratio and serum FFA concentration. In the clofibrate-treated rats there was no change in α- GP DAP ratio or α-GP concentration after ethanol, nor was there a significant rise in liver TG concentration. The initial serum TG concentration in the clofibrate-treated rats was about 30% of that in the normal animals. In the normal rats, ethanol administration produced a higher serum TG concentration than glucose, but in the clofibrate-treated rats it did not cause a significant rise in serum TG level. Thus, it appears that clofibrate treatment can inhibit the ethanol-induced change in the hepatic redox state in vivo as in the isolated liver. These results strengthen the view that clofibrate inhibits the production of acute ethanolic fatty liver at least in part by abolishing the ethanol-induced change in the hepatic redox state.

Effect of a transaminase inhibitor on the transport of cytosolic reducing equivalents into mitochondria.

Summary The effects of a transaminase inhibitor, cycloserine (CS), on extra and intramitochondrial redox state were studied in perfused rat liver. The addition of CS induced a rapid increase in perfusate lactate/pyruvate (L/P) ratio indicating a shift towards reduction in the cytosolic NAD(H) pool. The flavoprotein absorbance, measured from a lobe of liver, increased simultaneously indicating an oxidation of the intramitochondrial redox state. CS potentiated the effects of ethanol on the cytosolic redox state (L/P ratio), but decreased the ethanol-induced reduction of intramitochondrial flavo-proteins. The results can be interpreted to mean that transamination is of importance in the transport of reducing equivalents across the mitochondrial membrane.