A comparative study of the rate-limiting enzymes of cholesterol synthesis, esterification and catabolism in the alloxan-induced diabetic rat and rabbit

1. Hyperlipidaemia is associated with diabetes mellitus. 2. A comparison of cholesterol synthesis and utilization in alloxan-induced diabetic rats and rabbits revealed that interspecies differences existed only in the response of the key enzymes regulating cholesterol utilization, namely cholesterol 7 alpha-hydroxylase and ACAT in the liver and intestine respectively. 3. The activity of cholesterol 7 alpha-hydroxylase was enhanced in diabetic rats but was markedly reduced in diabetic rabbits. 4. Intestinal ACAT activity, though unchanged in diabetic rats was reduced in diabetic rabbits. 5. Such species differences in cholesterol utilization may underlie the different degree of susceptibility to hypercholesterolaemia that exists between these two species.     

Cretinism: influence on rate-limiting enzymes of amine synthesis in rat brain.

The effect of neonatal hypothyroidism on tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) activity, as well as on water content, was studied in different regions of the developing rat brain. Neonatal hypothyroidism, induced by daily treatment with propylthiouracil starting at birth, led to a cretinoid syndrome with a marked impairment of body and brain growth. Compared to control littermates, 30- and 45-day-old cretinous rats had elevated levels of water in the brain stem. The activities of TH and TPH were increased in a time-dependent manner in the brain stem, basal ganglia and hypothalamus of maturing cretinous animals. The increased activity of these rate-limiting enzymes of mono-amine synthesis may account for the elevated levels of brain norepi-nephrine and serotonin in rats subjected to neonatal hypothyroidism.     

Co-ordinate changes in enzymes of fatty acid synthesis, activation and esterification in rabbit mammary gland druing pregnancy and lactation.

1. The activities of fatty acid synthetase, acyl-CoA synthetase, glycerol phosphate acyltransferase and phosphatidate phosphatase were measured in the mammary glands of rabbits from day 16 of pregnancy to day 15 of post partum. 2. There were significant correlations between the increases in activities of these enzymes during this period. This was the case whether the activities were expressed per mg of homogenate protein, per g wet wt. of tissue or per total wet weight of the whole glands. The only exception was the lack of correlation between the activities of fatty acid synthetase and of phosphatidate phosphatase per g wet wt. of tissue. 3. These co-ordinate increases are discussed in relation to the changes which occur in fatty acid metabolism in the mammary gland during pregnancy and lactation.     

Transport of cholesterol into mitochondria is rate-limiting for bile acid synthesis via the alternative pathway in primary rat hepatocytes

Bile acid synthesis occurs mainly via two pathways: the "classic" pathway, initiated by microsomal cholesterol 7alpha-hydroxylase (CYP7A1), and an "alternative" (acidic) pathway, initiated by sterol 27-hydroxylase (CYP27). CYP27 is located in the inner mitochondrial membrane, where cholesterol content is very low. We hypothesized that cholesterol transport into mitochondria may be rate-limiting for bile acid synthesis via the "alternative" pathway. Overexpression of the gene encoding steroidogenic acute regulatory (StAR) protein, a known mitochondrial cholesterol transport protein, led to a 5-fold increase in bile acid synthesis. An increase in StAR protein coincided with an increase in bile acid synthesis. CYP27 overexpression increased bile acid synthesis by <2-fold. The rates of bile acid synthesis following a combination of StAR plus CYP27 overexpression were similar to those obtained with StAR alone. TLC analysis of (14)C-labeled bile acids synthesized in cells overexpressing StAR showed a 5-fold increase in muricholic acid; in chloroform-extractable products, a dramatic increase was seen in bile acid biosynthesis intermediates (27- and 7,27-hydroxycholesterol). High-performance liquid chromatography analysis showed that 27-hydroxycholesterol accumulated in the mitochondria of StAR-overexpressing cells only. These findings suggest that cholesterol delivery to the inner mitochondrial membrane is the predominant rate-determining step for bile acid synthesis via the alternative pathway.     

A comparative study of the conversion of 7-hydroxycholesterol in rabbit,guinea pig,rat, hamster,and chicken

The metabolism of epimeric 7-hydroxycholesterol was studied in vitro. 7Alpha-hydroxycholesterol or 7beta-hydroxycholesterol were incubated with rabbit, guinea pig, rat, hamster, and chicken microsomal suspensions and then extracted and analyzed using high-performance liquid chromatography (HPLC). 7Alpha-hydroxy-4-cholesten-3-one was the main product from 7alpha-hydroxycholesterol in the rabbit, guinea pig, and rat. A considerable amount of 7-ketocholesterol was also produced in the hamster and chicken. In all vertebrates, 7beta-hydroxycholesterol was converted only to 7-ketocholesterol in all vertebrates. 7Beta-hydroxy-4-cholesten-3-one was not detected. Reduction of 7-ketocholesterol was also studied in the rat and hamster. Whereas 7-ketocholesterol was converted to 7beta-hydroxycholesterol in the rat, it was converted to both 7alpha- and 7beta-hydroxycholesterol in the hamster. These results suggest that 7alpha-hydroxycholesterol is converted not only to 7alpha-hydroxy-4-cholesten-3-one but also to 7-ketocholesterol in the hamster and chicken. 7Beta-hydroxycholesterol was converted to 7-ketocholesterol in all vertebrates tested. The interconversion between 7alpha- and 7beta-hydroxycholesterol via 7-ketocholesterol was observed in the hamster in this in vitro study.     

Circadian rhythms of sterol 12alpha-hydroxylase, cholesterol 7alpha-hydroxylase and DBP involved in rat cholesterol catabolism

Circadian rhythms of important enzymes involved in the conversion of cholesterol to bile acids [sterol 12alpha-hydroxylase (12alpha-hydroxylase) and cholesterol 7alpha-hydroxylase (7alpha-hydroxylase)] and an albumin site D-binding protein (DBP) were examined in rats. When the animals were fed freely, they usually ate in the dark and the circadian rhythms of activities of 12alpha-hydroxylase and 7alpha-hydroxylase showed the same peaks (at 10 p.m.) and lows (at 2 p.m.). Their mRNA levels were determined at four timepoints: 3 a.m., 10 a.m., 3 p.m. and 10 p.m. A maximum of the rhythm of 12alpha-hydroxylase was observed at 3 p.m. and the minimum at 3 a.m. These results are distinct from those of 7alpha-hydroxylase, whose maximum point was at 10 p.m. and minimum at 3 p.m. When the rats were fed only in the day-time (from 9 a.m. to 5 p.m.), a marked shift of the activity and mRNA rhythms was observed with both enzymes. The circadian rhythms of the activities of both enzymes showed the same peaks (at 3 p.m.), but the mRNA levels of 12alpha-hydroxylase were distinct from those of 7alpha-hydroxylase, whose maximum point was at 3 a.m. and minimum at 10 p.m. Differences between the maximum and the minimum points of each enzyme mRNA level were statistically significant (P < 0.01 for 12alpha-hydroxylase and 0.05 for 7alpha-hydroxylase). Moreover, circadian rhythms of DBP were also markedly shifted with the change of feeding period. The maximum mRNA level was observed at 10 p.m. instead of 10 a.m. and the minimum was at 10 a.m. instead of 10 p.m.     

A comparative study on the peroxidase activity within the aqueous humor of the rabbit, guinea-pig and rat

Aqueous humor of the rabbit, guinea-pig and rat has been examined to determine if non-specific peroxidase activity is present within this ocular fluid. Substantial peroxidase activity was found within the aqueous humor of the rat. In contrast, no peroxidase activity could be detected in the aqueous humor of the rabbit or guinea-pig. Partial purification by gel chromatography revealed that the peroxidase within the aqueous humor of the rat has a mol. wt of approx. 45,000.     

Sterol balance studies in the rat. Effects of dietary cholesterol and beta-sitosterol on sterol balance and rate-limiting enzymes of sterol metabolism.

Sterol balance measurements using isotopic and chromatographic techniques were carried out in rats fed diets containing beta-sitosterol (0.8%) and cholesterol (1.2%). The activities of the rate-limiting enzymes of cholesterol synthesis (beta-hydroxy-beta-methylglutaryl-CoA reductase, EC 1.1.1.34) and bile acid synthesis (cholesterol 7 alpha-hydroxylase) were determined in the same animals. Cholesterol feeding increased cholesterol absorption from 1.2 to 70 mg/day. The increased absorption was compensated for by inhibition of hepatic cholesterol synthesis, enhanced conversion of cholesterol to bile acids (from 13.7 to 27.3 mg/day) and a slight increase in the excretion of endogenous neutral steroids (from 7.7 to 11.2 mg/day). Despite the adaptation there was accumulation of cholesterol in the liver (from 2.2 to 9.2 mg/g). Beta-Sitosterol feeding inhibited cholesterol absorption (calculated absorption was zero). In these rats there was enhanced cholesterol synthesis (from 20.0 to 28.8 mg/day, but no change in the rates of bile acid formation. Measurements of the activities of the rate-limiting enzymes showed fair correlation with cholesterol-bile acid balance. In cholesterol fed animals, beta-hydroxy-beta-methylglutaryl-CoA reductase was inhibited 80% and cholesterol 7 alpha-hydroxylase was enhanced 61%. In beta-sitosterol-fed animals, the reductase was increased 2-fold and cholesterol 7 alpha-hydroxylase was not significantly different from controls.     

Effect of catecholamines on the hepatic rate-limiting enzymes of cholesterol metabolism in normally fed and cholesterol-fed rabbits

Adrenergic control of liver cholesterol metabolism was studied in the rabbit. The effects of noradrenaline (α₁, α₂, β₂ agonist) and isoprenaline (β₁, β₂ agonist) on 3-hydroxy-3-methylglutaryl coenzyme A reductase, acyl-coenzyme A: cholesterol-o-acyltransferase (cholesterol acyltransferase) and cholesterol 7α-hydroxylase, the rate-limiting enzymes of cholesterol biosynthesis and esterification and bile acid synthesis, respectively, were examined in the normally fed and cholesterol-fed male New Zealand White rabbit. Isoprenaline increased the activities of hydroxymethylglutaryl CoA reductase and cholesterol acyltransferase approx. 12-fold and 5-fold, respectively, in normally fed rabbits. Noradrenaline, by contrast, produced an effect only on hydroxymethylglutaryl CoA reductase, the activity of which was increased 3-fold in these animals. Neither catecholamine had an effect on hydroxymethylglutaryl CoA reductase in the cholesterol-fed rabbit. Isoprenaline decreased the activity of cholesterol acyltransferase by approx. 40% and increased the activity of cholesterol 7α-hydroxylase 2-fold in the cholesterol-fed rabbit compared to cholesterol-fed controls. Noradrenaline had no effect on either cholesterol acyltransferase or cholesterol 7α-hydroxylase in either the normally fed or the cholesterol-fed rabbit. We suggest that β₂-adrenergic stimulation by isoprenaline in the normally fed rabbit may enhance cholesterol synthesis and storage, but that in the cholesterol-fed rabbit, it facilitates the elimination of cholesterol from the body by increasing the rate of bile acid synthesis.     

Enzymatic histamine catabolism in vertebrate ontogenesis. A comparative study

1. The synthesizing and degrading activities of histamine were determined in the liver and small intestine of developing guinea pig and chick embryos. 2. Though increasing with age, HDC values were always 2-3 orders of magnitude lower than those of degrading enzymes. 3. DAO activity on the other hand was 10-100 fold higher than HMT at all ages studied, suggesting a decisive role for oxidative deamination in control of tissue histamine levels. 4. Generally histamine levels were higher in tissues of developing guinea pig than chick embryo, however, in the laying hen intestine histamine concentration was approximately 5 times greater than in the adult guinea pig intestine.     

A comparative study on the integration of exogenous DNA into mouse, rat, rabbit, and pig genomes.

Transgenic mammals, from small laboratory rodents to domestic animals, have been successfully produced to date, but their production efficiency within or across species has been variable. This is probably due to the differences in the type of injected DNA and/or technical procedures employed in each laboratory, as well as the reproductive characteristics of the species. Here we report the direct comparison of the efficiencies of producing transgenic mice, rats, rabbits and pigs by one technician using a fusion gene composed of the bovine alpha S1-casein promoter and human growth hormone (hGH) gene. Before the fusion gene was injected into the zygotes, high magnitude centrifugation to visualize the pronuclei was necessary for all of the pig zygotes and one-third of the rabbit zygotes, but not for mouse and rat zygotes. Post-injection survival of the mouse zygotes (67.1%) was lower than those of the rat, rabbit and pig zygotes (89.6 to 100%). The volume change of the pronucleus following DNA injection was the lowest in mice (50% increase), moderate in rabbits (148% increase), and the most prominent in rats (238% increase). The data from only 1 pig zygote indicated a 22% increase in the pronucleus volume by DNA injection. The PCR analyses of the tail DNA of new born offspring indicated that 0.8% (4/493), 4.8% (22/463), 0.8% (3/367) and 0.9% (2/221) of the injected eggs in mice, rats, rabbits and pigs, respectively, developed into transgenic offspring. Some of the founder animals in all four species expressed the transgene in the mammary gland which was confirmed in hGH mRNA by RT-PCR and/or hGH peptide in Witch's milk with ELISA. These results suggest that the maximum volume of DNA solution injectable into the pronucleus is a possible factor explaining the species differences in the production of transgenic animals.     

Rates of cholesterol synthesis and low-density lipoprotein uptake in the adrenal glands of the rat, hamster and rabbit in vivo

The absolute rate of cholesterol acquisition from de novo synthesis and from receptor-dependent and receptor-independent low-density lipoprotein (LDL) uptake was determined in the adrenal glands of the rat, hamster and rabbit under in vivo conditions. The rate of incorporation of [3H]water into cholesterol in the adrenal gland was much higher in the hamster (1727 nmol/h per g) and rabbit (853 nmol/h per g) than in the rat (71 nmol/h per g). Assuming that 23 atoms of 3H are incorporated into the cholesterol molecule during its biosynthesis, the absolute rates of cholesterol synthesis were then calculated to equal 59, 29 and 2.4 micrograms/h per g of adrenal gland in the hamster, rabbit and rat, respectively. Rates of LDL-cholesterol uptake were measured using a primed continuous infusion of [14C]sucrose-labeled homologous LDL (total LDL transport) and methylated human LDL (receptor-independent LDL transport). The rate of total LDL-cholesterol uptake in the adrenal gland was much higher in the rabbit (227 micrograms/h per g) than in the rat (18 micrograms/h per g) or hamster (6 micrograms/h per g). In all three species LDL uptake was mediated largely (greater than 93%) by receptor-dependent mechanisms. In terms of total cholesterol acquisition, the hamster adrenal gland derived 10-times more cholesterol from de novo synthesis than from LDL uptake, whereas the converse was true in the rabbit. Rates of de novo synthesis and LDL-cholesterol uptake were both low in the rat adrenal gland, which is known to derive cholesterol mainly from circulating high-density lipoproteins. Thus, the adrenal gland acquires cholesterol for hormone synthesis from at least three different sources and the quantitative importance of these sources varies markedly in different animal species, including man.     

Role of aminopeptidases in enkephalin catabolism: comparative study of the regional distribution of aminopeptidases and enkephalinase A in the rat brain

In order to elucidate the role of aminopeptidases in enkephalin catabolism in rat brain, the local distribution of two types of cerebral cellular membrane aminopeptidases (puromycin-sensitive and puromycin-insensitive ones) and of the enkephalin system marker, enkephalinase A, was studied. It was found that the distribution patterns of the former enzymes differ essentially from that of enkephalinase A. Study of coupling between the enzymatic activities in different regions of rat brain revealed a strong correlation between the activities of puromycin-insensitive aminopeptidase and enkephalinase A in midbrain (including hypothalamus). It was supposed that in midbrain the role of aminopeptidase M in intrasynaptic inactivation of enkephalins is much more conspicuous than in other regions of rat brain. The puromycin-sensitive aminopeptidase activity does not seem to play a role in enkephalin catabolism.