Metabolic Regulation of Heme Catabolism and Bilirubin Production. I. HORMONAL CONTROL OF HEPATIC HEME OXYGENASE ACTIVITY Arne F. Bakken, … , M. Michael Thaler, Rudi Schmid J Clin Invest. 1972;51(3):530-536. https://doi.org/10.1172/JCI106841. Research Article Heme oxygenase (HO), the enzyme system catalyzing the conversion of heme to bilirubin, was studied in the liver and spleen of fed, fasted, and refed rats. Fasting up to 72 hr resulted in a threefold increase in hepatic HO activity, while starvation beyond this period led to a gradual decline in enzyme activity. Refeeding of rats fasted for 48 hr depressed hepatic HO activity to basal values within 24 hr. Splenic HO was unaffected by fasting and refeeding. Hypoglycemia induced by injections of insulin or mannose was a powerful stimulator of hepatic HO. Glucose given together with the insulin abolished the stimulatory effect of the latter. Parenteral treatment with glucagon led to a twofold, and with epinephrine to a fivefold, increase of hepatic HO activity; arginine, which releases endogenous glucagon, stimulated the enzyme fivefold. These stimulatory effects of glucagon and epinephrine could be duplicated by administration of cyclic adenosine monophosphate (AMP), while thyroxine and hydroxortisone were ineffective. Nicotinic acid, which inhibits lipolysis, failed to modify the stimulatory effect of epinephrine. None of these hormones altered HO activity in the spleen. These findings demonstrate that the enzymatic mechanism involved in the formation of bilirubin from heme in the liver is stimulated by fasting, hypoglycemia, epinephrine, glucagon, and cyclic AMP. They further suggest that the enzyme stimulation produced by fasting may be […] Find the latest version: https://jci.me/106841/pdf Metabolic Regulation of Heme Catabolism and Bilirubin Production I. HORMONAL CONTROL OF HEPATIC HEME OXYGENASE ACTIVITY ARNE F. BAKKEN, M. MICHAEL THALER, and RuDI ScEIMm From the Department of Medicine and Pediatrics, University of California, San Francisco, California 94122 A B S T R A C T Heme oxygenase (HO), the enzyme may be mediated by glucagon released in response to system catalyzing the conversion of heme to bilirubin, hypoglycemia. was studied in the liver and spleen of fed, fasted, and The possibility is considered that the enhanced HO refed rats. Fasting up to 72 hr resulted in a threefold activity in the liver may increase hepatic heme turn- increase in hepatic HO activity, while starvation be- over and hence, bilirubin production, which may ex- yond this period led to a gradual decline in enzyme plain the rise of unconjugated serum bilirubin observed activity. Refeeding of rats fasted for 48 hr depressed in fasting or hypoglycemic individuals. hepatic HO activity to basal values within 24 hr. Splenic HO was unaffected by fasting and refeeding. INTRODUCTION Hypoglycemia induced by injections of insulin or Clinical observations indicate that fasting leads to in- a of HO. mannose was powerful stimulator hepatic creased concentration of unconjugated serum bilirubin insulin the Glucose given together with the abolished in normal individuals (2), in patients with Gilbert's effect of Parenteral treatment stimulatory the latter. syndrome (3), and in normal horses (4). Spontaneous with glucagon led to a twofold, and with epinephrine hypoglycemia in newborn infants appears to aggravate of HO to a fivefold, increase hepatic activity; arginine, the transient hyperbilirubinemia of the neonatal period the en- which releases endogenous glucagon, stimulated In normal effects of and (5, 6). adults, hypoglycemia induced by in- zyme fivefold. These stimulatory glucagon fusion of mannose was found to be associated with epinephrine could be duplicated by administration of while transient unconjugated hyperbilirubinemia (7). cyclic adenosine monophosphate (AMP), thy- Although several possible mechanisms may be ad- were Nicotinic roxine and hydrocortisone ineffective. vanced to account for this positive relationship between acid, which inhibits lipolysis, failed to modify the stim- None of these hormones fasting or hypoglycemia and the rise in serum bilirubin, ulatory effect of epinephrine. a plausible explanation may be enhancement of bili- altered HO activity in the spleen. the mecha- rubin formation from increased heme turnover in the These findings demonstrate that enzymatic liver. This explanation is rendered particularly attrac- in the formation of bilirubin from heme nism involved tive because of analogous observations in hepatic por- in the liver is stimulated by fasting, hypoglycemia, epi- phyria, in which fasting stimulates and glucose ad- and further nephrine, glucagon, cyclic AMP. They sug- ministration represses formation of the heme precursors gest that the enzyme stimulation produced by fasting 8-aminolevulinic acid (8), porphobilinogen (8, 9), and This work was presented in part at the annual meeting protoporphyrin (10, 11) in the liver. of the American Association for the Study of Liver Dis- If increased bilirubin formation in the liver were to ease, Chicago, Ill. November 1970 (1). account for the hyperbilirubinemia of fasting, it would Dr. Bakken's present address is Department of Pediatrics, Rikshospitalet, University of Oslo, Oslo, Norway. be necessary to demonstrate (a) that the enzymatic Received for publication 10 May 1971 and in revised mechanism responsible for the conversion of heme to form 4 August 1971. bilirubin is stimulated by fasting, and (b) that this 530 The Journal of Clinical Investigation Volume 51 1972 enhanced enzyme activity increases hepatic heme catabo- lism which in turn, by the release of negative feedback repression (12, 13), would accelerate heme synthesis I z and turnover in the liver. of the first pos- _ Investigation o 4J tulate was made possible by the recent identification 6L 0 and characterization of microsomal heme oxygenase ? EL (HO)' (14, 15), which catalyzes the formation of bili- co D iz rubin in the liver. In the following experiments, HO _c activity was studied in the liver and spleen of rats z which had been fasted, rendered hypoglycemic, or treated with hormones that are related to hypoglycemia and fasting. METHODS HOURS FIGURE 1 Hepatic heme oxygenase activity in rats fasted Animals. In all experiments, female Sprague-Dawley up to 120 hr and in rats fasted for 48 hr and then refed rats of 200-300 g weight were used, except in one instance for 24 hr with laboratory chow and 5% glucose in drinking in which male rats of comparable weight were selected to water. Enzyme values are given as the mean and standard permit evaluation of the sex on the enzyme activity. Ani- deviation. The normal range of the enzyme activity in fed mals were maintained on standard chow and laboratory animals is indicated by the shaded area. water ad lib. Fasting rats were kept in individual cages with free access to water. Adrenalectomy or ovariectomy was performed under light cortisone acetate, Merck, Sharpe & Dohme, West Point, ether anesthesia. Operated animals and sham-operated con- Pa.), 10 mg; thyroxine (Na-levothyroxine, Synthroide, trols were used for the experiments 4-7 days after surgery. Flint Chemical Co., San Antonio, Texas), 10 mg; cyclic Adrenalectomized rats were given 0.9% aqueous saline in- AMP (adenosine 3',5'-cyclic monophosphoric acid, Sigma stead of drinking water. Chemical Co., St. Louis, Mo.), 1.25 mg; or dibutyryl cyclic Fasting and refeeding. Groups of rats were fasted for AMP (Ns02-dibutyryl adenosine 3',5'-cyclic monophosphoric various periods of time up to 120 hr before sacrifice for acid, Sigma Chem. Co.), 0.25 mg. In some animals a 10% enzyme assay. In all refeeding experiments, the fasting aqueous glucose solution was injected together with the was terminated after 48 hr, and the animals were refed epinephrine or glucagon, calculated to provide 0.5 g/kg per orally or by i.p. injections for 24 hr before being killed. hr of glucose for the 7 hr period before sacrifice. In 9 rats, For oral refeeding, the rats were offered laboratory chow epinephrine and glucagon were given simultaneously in the ad lib and drinking water containing 5% glucose. Intra- doses and at the times listed above. Epinephrine or glucagon peritoneal refeeding of fasted animals was performed by in the doses listed were also administered to rats that had two individual injections of 10% glucose in water, given been fasted for 48, 72, or 92 hr. The animals received two 24 and 8 hr before sacrifice. The glucose doses were chosen individual injections 7 and 5 hr before sacrifice while the to provide 0.5 g/kg per hr over this 24 hr refeeding period. food continued to be withheld. Induced hypoglycemia. Hypoglycemia was induced by re- Nicotinic acid (Matheson, Coleman & Bell, East Ruther- peated i.p. injections of insulin or mannose. Insulin (24 IU/ ford, N. J.) was administered i.p. in two individual in- mg, Sigma Chemical Co., St. Louis, Mo.) was given to jections in a dose of 10 mg per 100 g body weight 5 and fed rats in a dose of 1 or 12 IU per 100 g body weight 3 hr before sacrifice. In some instances 0.1 mg epinephrine 7 and 5 hr before sacrifice. In some experiments, 2 g of per 100 g body weight was given together with the nico- glucose per 100 g weight in 10%o aqueous solution was ad- tinic acid. Control animals received epinephrine alone at ministered together with the insulin. 5 and 3 hr. Four rats were injected with arginine (Sigma Mannose in 10%o aqueous solution was injected i.p. in fed Chem. Co.), in a dose of 50 mg per 100 g body weight animals 24 and 8 hr before sacrifice. The doses were se- 5 and 3 hr before killing. Control animals received 1 mg lected to provide 0.5 g/kg per hr of mannose for the 24 hr glucagon per 100 g body weight at the same schedule. period before the animals were killed. In another group Groups of adrenalectomized or ovariectomized rats were of rats, this regimen of mannose administration followed a fasted for periods of 48-72 hr.