Search

Growth hormone (GH) binding to its receptor modulates gene transcription by influencing the amount or activity of transcription factors. In the rat, GH exerts sexually dimorphic effects on liver gene transcription through its pattern of secretion which is intermittent in males and continuous in females. The expression of the CYP2C12 gene coding for the female-specific cytochrome P450 2C12 protein is dependent on the continuous exposure to GH. To identify the transcription factor(s) that mediate(s) this sex-dependent GH effect, we studied the interactions of the CYP2C12 promoter with liver nuclear proteins obtained from male and female rats and from hypophysectomized animals treated or not by continuous GH infusion. GH treatment induced the binding of a protein that we identified as hepatocyte nuclear factor (HNF) 6, the prototype of a novel class of homeodomain transcription factors. HNF-6 competed with HNF-3 for binding to the same site in the CYP2C12 promoter. This HNF-6/HNF-3 binding site conveyed both HNF-6- and HNF-3-stimulated transcription of a reporter gene construct in transient cotransfection experiments. Electrophoretic mobility shift assays showed more HNF-6 DNA-binding activity in female than in male liver nuclear extracts. Liver HNF-6 mRNA was barely detectable in the hypophysectomized rats and was restored to normal levels by GH treatment. This work provides an example of a homeodomain-containing transcription factor that is GH-regulated and also reports on the hormonal regulation of HNF-6.

In infants of diabetic mothers, maternal-fetal hyperglycemia induces fetal hyperinsulinemia, which may be sustained for several hours after birth. The inhibitory effect of insulin on glycogenolysis, gluconeogenesis, and lipolysis increases the risk of hypoglycemia in these infants. Eight term infants of diabetic mothers were studied between 3.9 and 8.5 h postnatally. The maternal diabetes was considered well controlled as judged by self-monitoring of blood glucose and Hb Alc. Neonatal plasma concentrations of glucose, glycerol, and insulin were monitored and averaged 2.7 +/- 0.7 mM, 371 +/- 116 microM, and 15.9 +/- 2.8 microU.mL-1, respectively. Stable isotope-gas chromatography/ mass spectrometry techniques were used to determine glucose and glycerol turnover rates and gluconeogenesis from glycerol in the infants. The appearance rates of glucose and glycerol averaged 20.0 +/- 5.4 mumol.kg-1.min-1 (3.6 +/- 1.0 mg.kg-1.min-1), and 8.9 +/- 2.3 mumol.kg-1.min-1, respectively. The fraction of glycerol appearance rate converted to glucose was 68.2 +/- 17.3%, which accounted for 15.5 +/- 4.6% of glucose production. Thus, compared with healthy term infants studied previously under identical conditions, the infants of diabetic mothers had higher insulin concentrations and attenuated glucose production. Despite increased insulin concentrations, lipolysis was unimpaired, and the gluconeogenic contribution from glycerol was higher than in the healthy newborns.

Autoimmune chronic active hepatitis (AI-CAH) is a feared component of autoimmune polyendocrine syndrome type I (APS I). In this study, immunoreactivity was assessed in sera from eight APS I patients, of whom three had AI-CAH, in an attempt to identify hepatic autoantigens. We performed indirect immunofluorescence staining of human and rat liver sections, Western blots on subcellular fractions of human and rat liver, immunoprecipitations of labelled aromatic L-amino acid decarboxylase (AADC) and cytochrome P450IA2 (CYP IA2) expressed by an in vitro transcription and translation system and studies of enzymatic activity. Autoantibodies against AADC were present in sera from all eight APS I patients, while immunoreactivity against CYP IA2 was only found in sera from the three APS I patients with AI-CAH. Enzymatic activity of CYP IA2 was inhibited by sera from APS I patients with AI-CAH but not by control sera. Our results show that CYP IA2 and AADC constitute hepatic autoantigens in patients with APS I and that immunoreactivity against CYP IA2 is associated with the presence of AI-CAH.

The newborn infant must mobilize endogenous substrate stores to meet the requirements of glucose-dependent organs. High concentrations of free fatty acids and glycerol, and a rapid decrease in the respiratory quotient, indicate that lipids are an important fuel soon after birth. The purpose of the present study was to determine the onset of lipolysis and gluconeogenesis from glycerol in healthy, term, unfed infants. Eight infants were studied from a postnatal age of 3.5 +/- 0.5 h to 7.4 +/- 0.2 h using [6,6-2H2] glucose and [2-13C]glycerol analysed by gas chromatography/mass spectrometry. Plasma concentrations of glucose, glycerol and insulin averaged 2.9 +/- 0.4 mM, 369 +/- 89 microM and 9.4 +/- 9.4 +/- 3.7 microU.ml-1, respectively. The hepatic glucose production rate averaged 25.0 +/- 3.5 mumol.kg-1 min-1 (4.5 +/- 0.6 mg.kg-1.min-1) and the endogenous plasma appearance rate of glycerol 8.7 +/- 1.2 mumol.kg-1.min. On average, 57.9 +/- 8.4% of the glycerol was converted to glucose, representing 11.1 +/- 2.3% of hepatic glucose output. Thus, lipolysis and gluconeogenesis from glycerol are established within the first 8 h of life in term infants.

Infantile Zellweger syndrome belongs to the group of peroxisomal disorders that lack peroxisomes. Both trihydroxycoprostanic acid (THCA), the precursor to cholic acid, and dihydroxycoprostanic acid (DHCA), the precursor to chenodeoxycholic acid, accumulate in this disease. In previous studies, we have shown that liver peroxisomes are required for the conversion of THCA into cholic acid both in vitro and in vivo by measuring a defective conversion in infants with Zellweger syndrome. In our present study, the conversion of DHCA into chenodeoxycholic acid has been measured in an infant with Zellweger syndrome to evaluate the importance of liver peroxisomes for the formation of chenodeoxycholic acid. Coprostanic acidemia was present from the second day of life with high levels of THCA and only trace amounts of DHCA. The conversion of i.v. administered [3H]DHCA into chenodeoxycholic acid was only 7% compared with the 80% conversion in an analogous study in an adult. There was, however, a rapid incorporation of 3H into biliary THCA and, after a lag phase, the 3H was incorporated into biliary cholic acid. After 72 h, 15% of [3H]DHCA was converted to cholic acid. The pool size of DHCA was 1.2 mg/m2 and the pool size of both cholic acid and chenodeoxycholic acid was markedly reduced. The renal excretion of cholic acid was more efficient than that of the less polar chenodeoxycholic acid. More polar metabolites of DHCA and THCA are formed in alternative metabolic pathways facilitating renal excretion of these toxic intermediates. We conclude that liver peroxisomes are essential for a normal conversion of DHCA into chenodeoxycholic acid.

The metabolism of 3 alpha, 7 alpha-dihydroxy-5 beta-cholestanoic acid was studied in the bile fistula rats and in preparations from rat liver homogenates. In the bile fistula rats, the main products were chenodeoxycholic acid, alpha-muricholic acid, and beta-muricholic acid. Only small amounts of cholic acid were formed. Incubations of 3 alpha, 7 alpha-dihydroxy-5 beta-cholestanoic acid with microsomes and NADPH yielded as the main product 3 alpha, 6 beta, 7 alpha-trihydroxy-5 beta-cholestanoic acid. The formation of small amounts of 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid was shown. The major product in incubations of 3 alpha, 7 alpha-dihydroxy-5 beta-cholestanoic acid with microsomes and the 100,000 g supernatant fluid fortified with ATP was identified as 3 alpha, 7 alpha, 24 xi-trihydroxy-5 beta-cholestanoic acid. This compound was converted into chenodeoxycholic acid and its metabolites in the bile fistula rat.

Conversion of 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid into 3 alpha, 7 alpha, 12 alpha 24-tetrahydroxy-5 beta-cholestanoic and cholic acids was catalyzed either by the mitochondrial fraction fortified with coenzyme A, ATP, MgCl2 and NAD or by the combination of microsomal fraction and 100,000 x g supernatant fluid fortified with coenzyme A, ATP and nad. 24-hydroxylation and formation of cholic acid occurred at similar rates with the 25R- and the 25S-forms of 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid. The 25R- and 25S-forms of 3 alpha, 7 alpha, 12 alpha-trihydroxy- and 3 alpha, 7 alpha, 12 alpha, 24-tetrahydroxy-5 beta-cholestanoic acids were administered to bile fistula rats. Labeled cholic acid was isolated from the bile. The initial specific radioactivity of cholic acid was higher and the disappearance of radioactivity more rapid after administration of 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid than of 3 alpha, 7 alpha, 12 alpha, 24-tetrahydroxy-5 beta-cholestanoic acid. The findings are discussed in relation to the assumed pathway for side chain cleavage in cholic acid biosynthesis.

Several hydroxylase activities in bile acid biosynthesis were assayed in subcellular fractions of human fetal liver. The livers were obtained at legal abortions performed between gestational weeks 14 and 24. Microsomal 12 alpha-hydroxylase and mitochondrial 12 alpha- and 26-hydroxylase activities were detected from week 14. The microsomal fraction also had capacity for 25-hydroxylation, whereas 7 alpha- and 26-hydroxylase activities were hardly detectable. The variation of the hydroxylase activities between different experiments can be explained by inactivation during the abortion or workup procedure. The results are discussed with respect to earlier studies of bile acid biosynthesis during development and adult life.

Metabolic pathways involved in the conversion of cholesterol to cholic and chenodeoxycholic acids have been investigated in bile fistula patients treated with a number of labeled potential bile acid intermediates. The findings of the present report indicate that the human liver cell has the capacity to synthesize both primary bile acids via multiple routes from cholesterol and 7 alpha-hydroxycholesterol. Evidence has been obtained for the existence of a major pathway to chenodeoxycholic acid via the 26-hydroxylation of 7 alpha-hydroxycholest-4-en-3-one. Cholic acid is synthesized preferentially via pathways from 5 beta-cholestane 3 alpha, 7 alpha-diol and a pathway from cholesterol not involving an initial 7 alpha-hydroxylation.

The specificity of the neonatal, andreogen-induced, irreversible programming of hepatic steroid metabolism in the rat was investigated. 5-alpha-Dihydrotestosterone propionate and estradiol benzoate were as efficient as testosterone propionate in inducing a male type of liver metabolism in the adult animal, whereas epitestosterone propionate, etiocholanolone propionate, and o,p'-DDT were practically inactive in this respect. These findings indicate that different mechanisms are involved in neonatal imprinting of hepatic steroid metabolism and in the well-known neonatal androgenic and estrogenic induction of persistent estrus and acyclic gonadotropin secreqion.

The presence of ectopic pituitary tissue (derived from an adult rat) in prepubertal male and female rats caused the immature, masculine-type hepatic steroid metabolism to develop into female-type metabolism. It is concluded that the hypothalamus-pituitary system controls the ontogenesis of sex-dependent steroid metabolism in rat liver.

The metabolism of tauro-[24-14C]deoxycholic acid was studied in microsomal preparations from fetal liver. The livers were obtained at legal abortions performed between week 13 and 24. Taurodeoxycholic acid was efficiently hydroxylated in the 1 beta- and 7 alpha-positions. The hydroxylase activities did not increase with gestational age. A marked variation in extent of hydroxylation was noted between different preparations. The results are discussed in relation to earlier knowledge of fetal and neonatal bile acid metabolism.

Conversion of 3alpha, 7alpha, 12alpha-trihydroxy-5beta-[7beta-3H]cholestanoic acid into 3alpha, 7alpha, 12alpha, 24-tetrahydroxy-5beta-cholestanoic acid in rat liver was catalyzed either by the mitochondrial fraction fortified with the 100,000 times g supernatant fluid or the microsomal fraction fortified with 100,000 times g supernatant fluid and ATP. The microsomal system was more active than the mitochondrial system. With the microsomal system the rate of reaction was considerably faster with free 3alpha, 7alpha, 12alpha-trihydroxy-5beta-cholestanoic acid as substrate than with the corresponding coenzyme A ester. Addition of coenzyme A inhibited the activity. Addition of cofactors other than ATP and coenzyme A did not markedly influence the reaction. The 100,000 times g supernatant fluid could be substituted with a protein fraction obtained by ammonium sulfate precipitation and Sephadex chromatography of the 100,000 times g supernatant fluid. The reaction was not catalyzed by a mixed function oxidase since there was no incorporation of 18O into the product when the reaction was performed in an atmosphere containing 18O2. On the other hand, oxygen may be obligatory since there was almost complete inhibition when the reaction was performed in an atmosphere consisting of nitrogen. Carbon monoxide did not inhibit the reaction. One atom of deuterium was incorporated into the product when the reaction was performed in a medium containing deuterated water. It was concluded that microsomal 24-hydroxylation of 3alpha, 7alpha, 12alpha-trihydroxy-5beta-cholestanoic acid involves the combined action of a desaturase and a hydratase. The reaction catalyzed by the hydratase appears to be stereospecific since the 24alpha epimer of 3alpha, 7alpha,12alpha-trihydroxy-5beta-cholestanoic acid was the predominant product. In contrast to the microsomal system, the mitochondrial system was not stimulated by the addition of ATP and was not inhibited by coenzyme A. The coenzyme A ester of 3alpha, 7alpha, 12alpha-trihydroxy-5beta-cholestanoic acid was 24-hydroxylated more efficiently than the free acid.

The effect of biliary obstruction on side chain hydroxylations in the biosynthesis and metabolism of bile acids was studied in the rat. For comparison, several other hydroxylation reactions in bile acid biosynthesis and metabolism were assayed. Biliary obstruction inhibited microsomal 26-hydroxylation of 5beta-cholestane-3alpha,7alpha-diol and microsomal 25- and 26-hydroxylation of 5beta-cholestane-3alpha,7alpha-12alpha-triol. Microsomal 7alpha-hydroxylation of cholesterol and 6beta-hydroxylation of lithocholic acid acid increased significantly, whereas the increase in microsomal 12alpha-hydroxylation of 5beta-cholestane-3alpha,7alpha-diol was less. Mitochondrial 26-hydroxylation of cholesterol, 5-cholestene-3beta,7alpha-diol, and 7alpha-hydroxy-4-cholesten-3-one was stimulated, whereas 26-hydroxylation of 5beta-cholestane-3alpha,7alpha-diol was not affected and that of 5beta-cholestane-3alpha,7alpha,12alpha-triol was markedly inhibited. The results indicate that mitochondrial 26-hydroxylation, particularly of substrates that primarily are precursors of chenodeoxycholic acid, plays a more important role in bile acid biosynthesis under conditions of biliary obstruction than under normal conditions.

The metabolism of [24-14C]lithocholic acid was studied in the microsomal fraction of fetal human liver homogenates. Fetal livers were obtained at legal abortions performed between wk 14 and 24. Product formation was analyzed by thin-layer chromatography, liquid chromatography, and gas chromatography-mass spectrometry. From wk 14 lithocholic acid was hydroxylated in several positions. Hyodeoxycholic acid (3 alpha,6 alpha-dihydroxy-5 beta-cholanoic) and 1 beta,3 alpha-dihydroxy-5 beta-cholanoic acid were identified among the products. The 3 beta-isomer of the former acid, 6 alpha-hydroxy-3-oxo-5 beta-cholanoic acid and a 2-hydroxylithocholic acid were tentatively identified. Two other metabolites carried a hydroxyl group in an unknown position, one of them probably at an angular methyl group. The highest total conversions per mg of microsomal protein were obtained with preparations from 18 wk fetuses. The results are discussed with particular reference to the role of lithocholic acid in fetal hepatotoxicity.