Co-cultures of hepatocytes with epithelial-like cell lines: Expression of drug-biotransformation activities by hepatocytes

To improve long-term expression of drug biotransformation activities in hepatocytes, we have examined the suitability of several epithelial-like cell lines (MDCK, MS and L-132) for supporting functional co-cultures with rat hepatocytes. Cells were selected on the basis of their compatibility with hepatocytes, formation of stable monolayers in the absence of serum and lack of drug biotransformation activities. The expression of individual elements of the biotransformation system was evaluated in these co-cultures. Co-cultured hepatocytes remained viable and showed a characteristic polygonal shape for more than a week. Depending on the cell line used, levels of aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O-deethylase activities of co-cultured hepatocytes oscillated between 24–47% of their initial value after 4 days in culture. The highest levels of monooxygenase activity were found in hepatocytes co-cultured with MS cells (41–47%). In contrast, these activities decreased to 6% when hepatocytes were maintained in pure culture for the same period. The activities of the conjugating enzymes UDP-glucuronyltransferase and glutathione S-transferase were maintained at nearly the initial levels during the complete period of study, both in pure and mixed-cultures, regardless of the cell line used. MS cells adapted themselves much better to serum-free culture conditions, and the co-culture with rat hepatocyte was technically easier. After one week, total cytochrome P450 and reduced glutathione in rat hepatocytes/MS co-cultures were 31% and 127% respectively of the day O values, whereas they were undetectable in pure culture. A clear induction of monooxygenase activities by methylcholanthrene, phenobarbital and ethanol could be observed by the 5th day in MS cells/hepatocyte co-cultures. The fact that the results of our work show the suitability of MS cells, an epithelial-derived cell line, for improving the expression of biotransformation enzymes of cultured hepatocytes opens new possibilities of simplifying co-cultures for their use in drug-metabolism studies.Abbreviations AHH aryl hydrocarbon hydroxylase - CDNB 1-chloro-2,4-dinitrobenzene - DMEM Dulbecco's modified Eagle's medium - ECOD 7-ethoxycoumarin O-deethylase - EDTA ethylenediamine tetraacetic acid - Et-OH ethanol - GSH reduced glutathione - GSH-t glutathione S-transferase - MC 3-methylcholanthrene - PB phenobarbital - UDP-Gt UDP-glucuronyltransferase     

Cpt1a silencing in AgRP neurons improves cognitive and physical capacity and promotes healthy aging in male mice

Orexigenic neurons expressing agouti-related protein (AgRP) and neuropeptide Y in the arcuate nucleus (ARC) of the hypothalamus are activated in response to dynamic variations in the metabolic state, including exercise. We previously observed that carnitine palmitoyltransferase 1a (CPT1A), a rate-limiting enzyme of mitochondrial fatty acid oxidation, is a key factor in AgRP neurons, modulating whole-body energy balance and fluid homeostasis. However, the effect of CPT1A in AgRP neurons in aged mice and during exercise has not been explored yet. We have evaluated the physical and cognitive capacity of adult and aged mutant male mice lacking Cpt1a in AgRP neurons (Cpt1a KO). Adult Cpt1a KO male mice exhibited enhanced endurance performance, motor coordination, locomotion, and exploration compared with control mice. No changes were observed in anxiety-related behavior, cognition, and muscle strength. Adult Cpt1a KO mice showed a reduction in gastrocnemius and tibialis anterior muscle mass. The cross-sectional area (CSA) of these muscles were smaller than those of control mice displaying a myofiber remodeling from type II to type I fibers. In aged mice, changes in myofiber remodeling were maintained in Cpt1a KO mice, avoiding loss of physical capacity during aging progression. Additionally, aged Cpt1a KO mice revealed better cognitive skills, reduced inflammation, and oxidative stress in the hypothalamus and hippocampus. In conclusion, CPT1A in AgRP neurons appears to modulate health and protects against aging. Future studies are required to clarify whether CPT1A is a potential antiaging candidate for treating diseases affecting memory and physical activity.     

Thermogenic effects of a "cafeteria" diet on the rat during its reproductive cycle

The heat production and oxygen consumption of intact virgin, pregnant, lactating and postlactating rats has been investigated both in groups fed a "cafeteria" diet as well as in controls. A third group of rats fed the cafeteria diet after parturition has been investigated. Pregnant rats fed a "cafeteria" diet increased their weight faster than controls. During lactation no increases in weight were observed, and in postlactation both "cafeteria" groups attained the same values higher than controls. The ingestion of a "cafeteria" diet resulted in higher heat production in all groups except lactating rats, which--in addition--showed higher heat outputs than all the other groups when the actual data were corrected by metabolic weight according to the surface law. The high lactation heat production (and oxygen consumption) can be a consequence of increased metabolic activity in the rat organism, devoted to milk production. It can be concluded that during lactation the dam energy output through the milk must absorb any increases in energy input due to the more densely-packed energy in the "cafeteria" diet, and this did not result in increased heat production.     

Biochemical functionality and recovery of hepatocytes after deep freezing storage

The present study was undertaken to define the conditions for optimal cryopreservation of hepatocytes. Two different freezing procedures were analyzed: a slow freezing rate (SFR) (-2 degrees C/min down to -30 degrees C and then quick freezing to -196 degrees C) and a fast freezing rate (FFR) (direct freezing of tubes to -196 degrees C: -39 degrees C/min). Cells were frozen in fetal bovine serum containing 10% Dimethyl sulfoxide (DMSO). After rapid thawing at 37 degrees C, followed by dilution and removal of the cryoprotectant, cells were plated and several parameters were followed as criteria for optimal cryopreservation of cells. The FFR cells showed no apparent ultrastructural damage after 24 h of culture. Plating efficiency and spreading were similar as controls. Gluconeogenesis from pyruvate and fructose, tyrosine amino transferase induction by glucagon and dexamethasone, urea production, and plasma protein synthesis of FFR cells were similar to those found in control cultures. The FFR procedure, in comparison to the SFR method, seemed to render the best preserved hepatocytes.     

Plasma clearance, organ distribution and target cells of interleukin-6/hepatocyte-stimulating factor in the rat

The plasma half-life of recombinant human interleukin-6 (rhIL-6) was determined in rats by measuring the disappearance of the biological activity as well as of the radioactivity of 125I-rhIL-6 from the circulation. The kinetics of clearance were biphasic. It consisted of a rapid initial disappearance corresponding to a half-life of 3 min, and of a second slow one corresponding to a half-life of about 55 min. By cellulose-acetate electrophoresis it was shown that rhIL-6 binds to a plasma protein resulting in a complex migrating in the beta-gamma region; 20 min after intravenous injection, about 80% of the 125I-rhIL-6 that had disappeared from the circulation was found in the liver. 125I-rhIL-6 was exclusively localized on the surface of parenchymal cells suggesting the existence of an interleukin-6 receptor on the hepatocytes.     

Effect of chronic inflammation on copper and zinc metabolism

The effect of chronic inflammation on serum, blood and hepatic copper and zinc concentrations has been studied in arthritic female Sprague-Dawley rats, for a six week period. Metals were determined by atomic-absorption spectrophotometry and by inductively-coupled argon plasma spectrometry. Blood measurements evidenced hypercupremia and normal zinc levels. Serum and hepatic copper content were found to be increased. Serum zinc values were reduced whilst hepatic zinc concentration was higher. Serum alterations of both metals remained throughout the studied period. Good correlations were found between systemic alteration and changes in metal values.     

Presence of chloride cells in gill filaments and lamellae of the skate Torpedo marmorata

The morphology of chloride cells in the gill of the skate Torpedo marmorata is described from a light and an electron microscopic study. Chloride cells have been regularly observed in both the gill filament (or primary epithelium) and the lamellae (or secondary epithelium). Chloride cells located in the filament usually display convex apical regions with microvilli protruding amongst microridges of neighbouring pavement cells, whereas chloride cells in the lamellae are located between the two epithelial layers, and contact with the external milieu is via a narrow apical opening. Present observations are discussed in relation to data on the presence of chloride cells in the lamellae of marine teleost fish, and it is suggested that the occurrence of chloride cells all along the lamellae might be in some way inversely related to fish activity.     

Effects of cholinergic stimulation on human esophageal peristalsis.

The effects of cholinergic stimulation on esophageal peristalsis were studied in normal subjects. An intraesophageal transducer assembly was used to measure the dynamics of esophageal peristalsis before and after edrophonium chloride, 80 mug/kg intravenously. Following endrophonium, there was a marked increase in amplitude of esophageal persistalsis associated with a significant reduction in velocity of esophageal peristalsis and a significant increase in width and time of appearance of the contraction wave following a swallow. Graded doses of endrophonium all resulted in significant increases in peristaltic amplitude with the maximal response occurring at doses of 80 and 160 mug/kg. Similar results were obtained with a more direct-acting cholinergic agent, bethanechol, 80 mug/kg subcutaneously. The relevance of these results as an indication of the importance of cholinergic innervation in regulating esophageal motility are discussed.     

Fetal sex determination by ultrasonically locating the genital tubercle in ewes

Accuracy of transrectal ultrasonographic determination of fetal sex by identifying and locating the genital tubercle was determined in 29 ewes with single fetus at 60 to 69 d post breeding. Examinations were conducted once under farm conditions utilizing a real-time diagnostic scanner equipped with a linear-array 5 MHz transducer. Definite diagnoses were made after lambing. Total accuracy was 89%, and 100% (14/14) of the male fetuses and, 76% (10/13) of the female fetuses were correctly diagnosed. In 2 (7%) ewes, fetal sex was not determinable.