Rainer N. Zahlten

The isolation of hormone-sensitive rat hepatocytes by a modified enzymatic technique

Abstract Hepatocytes that are similar to the perfused liver in glucagon sensitivity can be obtained in a high, reproducible yield by modifications of the well-known enzymatic technique for the preparation of isolated liver cells. The major modifications are: (a) a simple, economic, and temperature-controlled apparatus for the recirculating perfusion of the isolated rat liver; (b) the use of substrate-fortified calcium-free Krebs-Henseleit bicarbonate buffer; and (c) high perfusion rates, which lead to the isolation of hepatocytes with normal ultrastructure and metabolic activities. From 4 × 10 8 to 5 × 10 8 cells can be routinely isolated from an 8- to 10-g liver independent of the collagenase preparations applied. The rat liver cells are viable (90–95%) by various criteria including electron microscopy and exclusion of 0.2% trypan blue. When studying various incubation techniques, it was observed that the use of gelatin in the medium is preferred as compared to albumin Fraction V or fatty acid-free albumin which tended to inhibit gluconeogenic rates from various substrates in calcium-free medium. Addition of calcium chloride to the incubation medium strikingly improved gluconeogenesis from lactate. Various procedures for calculating the number of cells corresponding to 1 g wet liver tissue are discussed in detail.

The influence of ammonium and calcium ions on gluconeogenesis in isolated rat hepatocytes and their response to glucagon and epinephrine

Abstract The use of n-butylmalonate as an inhibitor of malate transport from mitochondria and of aminooxyacetate as an inhibitor of glutamate-aspartate transaminase indicated that rat liver hepatocytes employ the aspartate shuttle for gluconeogenesis from lactate which supplies reducing equivalents to the cytosolic NAD system. In contrast, malate is transported from mitochondria to cytosol for gluconeogenesis from pyruvate. This conclusion is corroborated by the finding that the addition of ammonium ions enhances gluconeogenesis from lactate but inhibits glucose formation from pyruvate. In hepatocytes, glucagon and epinephrine have relatively little effect on glucose synthesis from lactate. Ammonium ions permit both of these hormones to exert their usual stimulation of gluconeogenesis from lactate. Calcium ions (1.3 m m ) enhance gluconeogenesis from lactate and from lactatepyruvate mixtures (10:1). The stimulatory effects of Ca2+ and NH4+ are additive and, when lactate is the substrate, the rates of gluconeogenesis achieved are so high as to preclude further stimulation by glucagon.

Pyruvate uptake in rat liver mitochondria: Transport or adsorption?

Studies were performed in our laboratory to investigate certain aspects of pyruvate translocation into rat liver mitochondria, which is still a matter of controversy. Even though evidence has been collected in recent years showing that monocarboxylate uptake in mitochondria is not a controlled and carrier-limited translocation [ 1, 21, Papa et al. [3] reported recently that pyruvate is transported through a specific translocator into mitochondria. The method selected for our study was the technique of rapid centrifugation (with or without silicone layer), which was established as the method of choice for many carboxylic acid [l] and adenine nucleotide [ 1,4] transport and exchange studies. Mitochondrial terminology such as “uptake” and “transport” (translocation of a substrate from one side of the membrane to the other) usually does not distinguish precisely between a translocation through a membrane system or a simple “surface limited” or “bulk phase limited” [5] adsorption of anions or cations on proteins and lipids. For these transport studies, we prepared different denatured, metabolically and structurally destroyed mitochondria as a control to normal mitochondria. We present evidence that it is not possible to justify the kinetic treatment [3] of the data obtained with the method of rapid centrifugation as representing active transport. Adsorption of pyruvate to mitochondrial proteins and/or lipids rather than specific transport to the matrix space appears to account for the binding of pyruvate by rat liver mitochondria.

Artifacts in protein synthesis by mitochondria in vitro.

Protein synthesis by mitochondria in viva and irl vitro has been the subject of an increasing number of publications in recent years [ 1,2]. The importance of the mechanism of mitochondrial biogenesis shifted most investigations from the more complex ill vivo studies to isolated in vitro systems. One of the major disadvantages of using isolated mitochondria is the very low incorporation rate of amino acids into the few mitochondria proteins which can be coded for by mitochondrial DNA. As we will show in this paper, there is striking evidence that despite an apparent regulated protein synthesis (temperature-, concentration-, and time-dependency) the “incorporation” of certain amino acids into mitochondrial proteins in vitro reflects rather specific binding to, or chemical interactions with, the mitochondrial protein-lipid structures. Our amino acid “incorporation” rates are quantitatively comparable to others published previously from various laboratories as reflecting true mitochondrial protein synthesis. The chemical basis of the described phenomena will be discussed.