Hepatic zonation of carbon and nitrogen fluxes derived from glutamine and ammonia transformations

Journal of Biomedical Science RHeseeaprchatic zonation of carbon and nitrogen fluxes derived from glutamine and ammonia transformations Jurandir F Comar Fumie Suzuki-Kemmelmeier Jorgete Constantin Adelar Bracht 0 Laboratory of Liver Metabolism, Biochemistry Department, University of Maringa , Maringa , Brazil Background: Glutaminase predominates in periportal hepatocytes and it has been proposed that it determines the glutamine-derived nitrogen flow through the urea cycle. Glutamine-derived urea production should, thus, be considerably faster in periportal hepatocytes. This postulate, based on indirect observations, has not yet been unequivocally demonstrated, making a direct investigation of ureogenesis from glutamine highly desirable. Methods: Zonation of glutamine metabolism was investigated in the bivascularly perfused rat liver with [U14C]glutamine infusion (0.6 mM) into the portal vein (antegrade perfusion) or into the hepatic vein (retrograde perfusion). Results: Ammonia infusion into the hepatic artery in retrograde and antegrade perfusion allowed to promote glutamine metabolism in the periportal region and in the whole liver parenchyma, respectively. The results revealed that the space-normalized glutamine uptake, indicated by 14CO2 production, gluconeogenesis, lactate production and the associated oxygen uptake, predominates in the periportal region. Periportal predominance was especially pronounced for gluconeogenesis. Ureogenesis, however, tended to be uniformly distributed over the whole liver parenchyma at low ammonia concentrations (up to 1.0 mM); periportal predominance was found only at ammonia concentrations above 1 mM. The proportions between the carbon and nitrogen fluxes in periportal cells are not the same along the liver acinus. Conclusions: In conclusion, the results of the present work indicate that the glutaminase activity in periportal hepatocytes is not the rate-controlling step of the glutamine-derived nitrogen flow through the urea cycle. The findings corroborate recent work indicating that ureogenesis is also an important ammonia-detoxifying mechanism in cells situated downstream to the periportal region. - Background Glutamine is one of the most abundant amino acids in the organism of mammals and it is involved in more metabolic processes than any other amino acid [1]. Also for the liver the role of glutamine is very important. It is known that the metabolism of glutamine presents zonation [2], i.e., the different regions along the hepatic acini respond in a different way to the amino acid [3-5]. The underlying mechanisms of the zonation of enzymes involved in glutamine metabolism are controversial. Recent work has suggested a role for levels of substrate, autocrine soluble factor or cytoskeleton interactions putatively associated with the beta-catenin signaling pathway [6]. Knowledge about zonation of the metabolism of L-glutamine is centered mainly on nitrogen metabolism. The dominant idea is that, along the hepatic acinus, the pathways of urea production and glutamine synthesis are arranged in sequence in order to optimize ammonia detoxification. The urea synthesis in the periportal region represents the system of low affinity for ammonia detoxification. Glutamine synthesis in the perivenous zone represents the system of high affinity for ammonia detoxification. The periportal glutaminase [7], located in the mitochondria, is stimulated by ammonia and influenced by pH and hormones [8,9]. The activity of this enzyme is believed to determine, partly at least, the flow of nitrogen derived from glutamine through the urea cycle [10]. The glutamine synthetase, restricted to a limited number of perivenous hepatocytes, is believed to act as a kind of scavenger for the ammonia that escapes from the periportal urea synthesis [3]. If the activity of glutaminase determines the nitrogen flow derived from glutamine through the urea cycle, urea production from glutamine should be considerably faster in periportal hepatocytes [5,10]. This postulate is based on the measurement of enzymatic activities under artificial conditions and has not yet been unequivocally demonstrated by flux measurements in intact cell systems. Such measurements are important, however, because discrepancies between enzyme activity or gene expression evaluations and the actual metabolic fluxes in the living cell are common. Recent studies have shown, for example, that urea production from alanine, lactate + ammonia and pyruvate + ammonia is faster in cells situated downstream to the periportal zone at most substrate concentrations [11,12] in spite of the observation that the expression of key enzymes from the urea cycle predominates in these cells [13]. Periportal predominance of urea production was found only at high ammonia concentrations in the presence of pyruvate [12]. Absence of correlation between enzyme activity or enzyme expression and metabolic fluxes in the cell are actually quite common and direct measurements of the latter are, thus, desirable. Moreover, glutamine is also a gluconeogenic substrate and it has been found that periportal and perivenous cells present different glucose to urea production ratios from alanine [11]. This is an important observation if one takes into account the reciprocal regulation of both ureogenesis and gluconeogenesis [14,15], which seems to be different in periportal and perivenous cells, and raises the question about the relative proportions between ureogenesis and glutamine transformation. These and other questions prompted us to undertake a detailed investigation of the zonation of glutamine transformation with the simultaneous measurement of nitrogen and carbon fluxes. The methodology to be utilized is the bivascularly perfused rat liver, which allows to reach selectively periportal hepatocytes via the hepatic artery in retrograde perfusion [16] and which has been successfully used for investigating hepatic zonation without significant alterations of the liver structure [11,12,17]. Methods Materials The liver perfusion apparatus was built in the workshops of the University of Maring. Enzymes and coenzymes used in the metabolite assays were purchased from Sigma Chemical Co. (St Louis, USA). [U-14C]Glutamine (258 mCi/mol) was purchased from Amersham Bioscience (Buckimghamshire, UK). All standard chemicals were from the best available grade (>99.5% purity) and were purchased from Merck (Darmstadt, FRG), Carlo Erba (So Paulo, Brasil) and Reagen (Rio de Janeiro, Brazil). Animals and bivascular liver perfusion Male albino rats (Wistar), weighing 180-220 g, were fed ad libitum with a standard laboratory diet (Purina). Food was withdrawn 18 hours prior to the liver perfusion experiments. For the surgical procedure of liver isolation, the rats were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/kg). All experiments were done in accordance with the world-wide accepted ethical guidelines for animal experimen (...truncated)