A new method for the isolation of fresh hepatocytes from periportal and pericentral regions of the liver lobule

Misra, U. K.; Yamanaka, Hirofumi; Kizaki, Zenro; Kauffman, F. C.; Thurman, Ronald G.

doi:10.1016/s0006-291x(88)81108-2

Cited by 11 publications

(3 citation statements)

References 16 publications

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“…The heterogeneous population of freshly isolated hepatocytes were cultured for 48 h with either high glucagon (10 nmol/1) levels to obtain periportal-like cells or with high insulin (10 nmol/1) levels to obtain perivenous-like cells. After a medium change, enzyme activities (pmol X min"1 X mg DNA-1) and metabolic rates (pmol Q unit X h-1 X mg DNA-1) were determined under substrate condi tions mimicking the postabsorptive (5 mmol/1 glu ences between the two cell populations in the relative utilization of gluconeogenic precur sors and glucose were dependent on the pres ence of insulin and on the nutritional state of the rat [42], Finally, the rate of oxygen up take, which can be linked functionally to glu coneogenesis, was higher in periportal than in perivenous plugs obtained by a micro punch method from perfused rat liver [63].…”

Section: Fluxes In Vitro Studied With Invasive Techniquesmentioning

confidence: 98%

Hepatocyte Heterogeneity in the Metabolism of Carbohydrates

Jungermann

Thurman²

1992

Enzyme

109

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Periportal and perivenous hepatocytes possess different amounts and activities of the rate-generating enzymes of carbohydrate and oxidative energy metabolism and thus different metabolic capacities. This is the basis of the model of metabolic zonation, according to which periportal cells catalyze predominantly the oxidative catabolism of fatty and amino acids as well as glucose release and glycogen formation via gluconeogenesis, and perivenous cells carry out preferentially glucose uptake for glycogen synthesis and glycolysis coupled to liponeogenesis. The input of humoral and nervous signals into the periportal and perivenous zones is different; gradients of oxygen, substrates and products, hormones and mediators and nerve densities exist which are important not only for the short-term regulation of carbohydrate metabolism but also for the long-term regulation of zonal gene expression. The specialization of periportal and perivenous hepatocytes in carbohydrate metabolism has been well characterized. In vivo evidence is provided by the complex metabolic situation termed the ‘glucose paradox’ and by zonal flux differences calculated on the basis of the distribution of enzymes and metabolites. In vitro evidence is given by the different flux rates determined with classical invasive techniques, e.g. in periportal-like and perivenouslike hepatocytes in cell culture, in periportal- and perivenous-enriched hepatocyte populations and in perfused livers during orthograde and retrograde flow, as well as with noninvasive techniques using miniature oxygen electrodes, e.g. in livers perfused in either direction. Differences of opinion in the interpretation of studies with invasive and noninvasive techniques by the authors are discussed. The declining gradient in oxygen concentrations, the decreasing glucagon/insulin ratio and the different innervation could be important factors in the zonal expression of the genes of carbohydrate-metabolizing enzymes. While it is clear that the hepatocytes sense the glucagon/insulin gradients via the respective hormone receptors, it is not known how they sense different oxygen tensions; the O(2) sensor may be an oxygen-binding heme protein. The zonal separation of glucose release and uptake appears to be important for the liver to operate as a ‘glucostat’. Thus, zonation of carbohydrate metabolism develops gradually during the first weeks of life, in part before and in part with weaning, when (in rat and mouse) the fat- and protein-rich but carbohydrate-poor nutrition via milk is replaced by carbohydrate-rich food. Similarly, zonation of carbohydrate metabolism adapts to longer lasting alterations in the need of a ‘glucostat’, such as starvation, diabetes, portocaval anastomoses or partial hepatectomy.

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Section: Fluxes In Vitro Studied With Invasive Techniquesmentioning

confidence: 98%

Hepatocyte Heterogeneity in the Metabolism of Carbohydrates

Jungermann

Thurman²

1992

Enzyme

109

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“…Future developments of the cell sort ing technique based on various forms of functional in vivo labelling holds significant promise [42], but appear to be limited in the cell yield/stability of the preparation during isolation [43]. In this context, the technique of Misra et al [44], isolating cell plugs from the PP and PV zones on the liver surface, is very interesting, although it seems to be lim ited both in terms of yield and by the same problems which complicate the experiments with tissue slices. The digitonin-pulse perfu sion adds an important new feature by allow ing very rapid sampling of PP and PV cyto sol with high zonal selectivity.…”

Section: Resultsmentioning

confidence: 99%

Hepatocyte Heterogeneity in the Metabolism of Fatty Acids: Discrepancies on Zonation of Acetyl-CoA Carboxylase

Quistorff

Katz

Witters³

1992

Enzyme

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Lipid metabolism appears to be less zonated than carbohydrate and protein metabolism. Studies on the zonation of lipid metabolism have been centered in particular on fatty acid synthesis which, according to the concept of metabolic zonation, should be a predominantly perivenous process while fatty acid oxidation should be periportal. There are, however, conflicting data on the activity gradients of lipogenic enzymes as well as measurements of actual synthesis of fatty acid and very low density lipoprotein. Data obtained by microdissection show a 1.5- to 2-fold higher activity of acetyl-CoA carboxylase and citrate lyase in the perivenous zone in agreement with measurements of the actual rate of fatty acid synthesis in preparations of hepatocyte, enriched in periportal or perivenous cells. On the other hand, results obtained with the dual-digitonin-pulse perfusion technique demonstrate the opposite gradient in the form of a 2- to 3-fold higher specific activity of acetyl-CoA carboxylase in the periportal zone based on measurements of the acetyl-CoA carboxylase protein proper. This specific activity gradient, which applies to male and not female rats, disappears almost completely in the fasted-refed animal, were lipogenesis is strongly induced. In this review we attempt to rationalize these discrepancies in the results as methodological differences which in particular apply to the following parameters: (1) expression of results (reference substance); (2) selectivity of zonal sampling, and (3) differences in methodology of acetyl-CoA carboxylase measurements. It is concluded that these factors could account for the discrepancies, but further studies, in particular on the zonation acetyl- CoA carboxylase mRNA, are required in order to further understand the zonation of lipid metabolism and its possible role in the metabolic regulation of the liver.

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“…The digitonin/collagenase technique described by Lindros and Penttila (1985) and Quistorff (1985) appears to be the most successful, although these methods generally result in preparations of low initial viability (Burger et al, 1989). Isolation of periportal and perivenous hepatocytes has also been achieved by microdissection (Misra et al, 1988), but this is tedious and generates low yields.…”

Section: Introductionmentioning

confidence: 99%

Periportal- and perivenous-enriched hepatocyte couplets: differences in canalicular activity and in response to oxidative stress

Wilton

Chipman

Lawson

et al. 1993

Biochemical Journal

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Unlike isolated single hepatocytes, hepatocyte couplets retain their apical polarity, and, during short-term culture form an enclosed canalicular space or vacuole between the two adjacent cells into which biliary secretion is initiated. Hepatocyte couplets were prepared after partial collagenase perfusion of rat liver. Centrifugal elutriation was used to fractionate the preparation into six couplet-containing suspensions. Image analysis was used to determine the size of cultured couplets. The size of the couplets ranged from 34.1 +/- 0.76 microns and 684 +/- 24.1 microns 2 (mean length and area respectively +/- S.E.M.) in Fraction 2, to 43.7 +/- 0.57 microns and 1033 +/- 33.8 microns 2 length and area respectively in Fraction 7. Glutamine synthetase activity was assessed in each freshly eluted fraction and was shown to be predominant in Fractions 6 and 7. Pretreatment of rats with CCl4, which selectively destroys perivenous hepatocytes, decreased the proportion of couplets in these fractions by over 67%, and their glutamine synthetase activity by over 97%. It was concluded that Fractions 2 and 3 contained predominantly couplets of Zone 1 (periportal) origin, Fractions 4 and 5 those from Zone 2, and Fractions 6 and 7 predominantly couplets of Zone 3 (perivenous) origin. The development of canalicular secretory activity was assessed in the couplets after a 15 min incubation with a fluorescent bile acid, cholyl-lysyl-fluorescein (CLF). This was sigmoidal in all fractions, but slower in the periportal couplets, taking 5.1 h for 50% to show secretory activity in Fraction 2, compared with 2.7 h for Fraction 7. Incubation of hepatocyte couplets with 1 or 10 microM taurodehydrocholate, a non-toxic bile acid analogue, did not influence the rate of development of accumulation of CLF by the couplets or the area of the canalicular vacuole in any fraction. However, it did decrease the CLF content of couplets incubated with CLF for 15 min to a greater extent in those of perivenous origin. After subjecting the couplets to oxidative stress by incubation with 20 microM menadione (2-methyl-1,4-naphthoquinone), it was evident that periportal couplets were less able to maintain canalicular secretory activity than perivenous couplets.

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A new method for the isolation of fresh hepatocytes from periportal and pericentral regions of the liver lobule

Cited by 11 publications

References 16 publications

Hepatocyte Heterogeneity in the Metabolism of Carbohydrates

Hepatocyte Heterogeneity in the Metabolism of Carbohydrates

Hepatocyte Heterogeneity in the Metabolism of Fatty Acids: Discrepancies on Zonation of Acetyl-CoA Carboxylase

Periportal- and perivenous-enriched hepatocyte couplets: differences in canalicular activity and in response to oxidative stress

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