Characterization of Gluconeogenesis in Enzymatically Isolated Parenchymal Cells of Rat Liver

Abstract

Evaluation of hepatic paren'chyma.1 cell metabolic functions in vitro has been attempted utilizing a number of test systems, viz., the perfused liver (1, 2) liver slices (3, 4), andl liver cells isolated by a variety of techniques (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). While studies of the perfused rat liver have been extensive and successful, two1 major disadvantages of this system are (i) it is not exclusively a population of parenchymal cells, and (ii) the number of variables able to be studied simultaneously is limited. The value of liver slices is also restricted since they are a nonuniform and complex system in which oxygen diffusion to cells on the inside of the slice is limited and cells on the outer surface are mechanically damaged. A homogeneous preparation od intact parenchymal cells overcomes many of the disadvantages mentioned in the other systems and, in addition, permits the study of many variables while also providing simultaneous control samples.The purpose of the present investigation was to characterize a method for the preparation of intact viable hepatocytes with particular emphasis on their application to the in ;Jitro study of gluconeogenesis. The method employed was a modification of the enzymatic technique developed by Berry and Friend (6) and involved perfusion of isolated rat livers with media containing collagenase and hyaluronidase. The ability of the parenchymal cells isolated by this method to exclude vital dyes and to retain enzymes localized to the cytoplasmic, mitochondrial, and lysosomal portions of the cell was deter-mined. These tests were deemed critical because mechanically isolated hepatocytes have been shown to be demaged sufficiently to take up trypan blue and to leak cytoplasmfic enzymles (5, 9, 11). Since Krebs and coworkers ( 2 ) have stated that a stringent test of the metabolic integrity of the hepatocyte is its ability to synthesize glucose from lactate, the gluconeogenic capacity of the isolated cells was evaluated in the present study. Of special interest was the inlfluence of fasting duration on hepatocyte gluconeogenesis in light of recent evidence identifying peripheral substrate release as the probable rate controllling factor in hepatic gluconeogenesis during prolonged !fasting (116). Materials and Methods. A n h a b .Male rats of the Holtzman strain (Holtzman Co., Madison, WI) weighing 280-310 g were fed Purina chow and water ad libitum in animal quarters maintained a t 76-78' F and at an automatically regulated 12-hr light-dark cycle (7 AM-7 P M ) . Experiments commenced a t 9 AM on rats which were fasted with access to water for 24, 48, 96, or 168 hr prior to use.Liver isolation. Parenchymal cells were isolated from livers by a modification of the method of Berry and Frliend (6). Rats were anesthetized with sodium pentobarbital a t a dose of 30 mg/kg via the dorsal vein of the penis. A midline laporotomy was performed, the inferior vena cava and portal vein were isolated, and 500 USP units of heparin were injected iv. After ligating the abdominal vena ...

Bile salt transport after selective damage to acinar zone 3 hepatocytes by bromobenzene in the rat

“…Cells were examined prior to the incubation by light microscopy with and without the addition of trypan blue, as described previously (Gumucio et al, 1978b). Gluconeogenesis was assayed according to Cornell and Filkins (1974), using 10 rnM lactate as substrate. Glucose produced during the 2 hr of incubation was measured by the glucose oxydase method (Statzyme glucose, Worthington Biochemical).…”