W.J. Vaartjes

Inhibition by fatty acyl esters of adenine nucleotide translocation in rat-liver mitochondria.

At low substrate concentrations linoleate oxidation in isolated rat-liver mitochondria can be stimulated by addition of phosphate and phosphate acceptor. With ADP as phosphate acceptor this State-3 linoleate oxidation is strongly inhibited after addition of CoASH. Inhibition by CoASH itself seems rather unlikely. However, in media containing ADP and Mg²+ the longchain acyl-CoA synthetase present in the outer mitochondrial membrane can form linoleyl-CoA, ATP being derived from ADP through the action of adenylate kinase.

Phorbol esters, but not epidermal growth factor or insulin, rapidly decrease soluble protein kinase C activity in rat hepatocytes.

Exposure of freshly isolated rat hepatocytes to tumor-promoting phorbol esters like phorbol 12-myristate 13-acetate resulted in a time- and concentration-dependent translocation of protein kinase C from the soluble to the particulate fraction of the cells. No such disappearance of soluble protein kinase C activity was observed with either epidermal growth factor or insulin, indicating that activation of protein kinase C is not necessarily involved in the short-term metabolic action of physiological growth factors on rat hepatocytes.

Regulation of pyruvate metabolism by the mitochondrial energy state: The effect of palmityl-coenzyme A

In isolated rat liver mitochondria pyruvate can either be decarboxylated or carboxylated. In the former case the pyruvate dehydrogenase complex (EC 1.2.4.1) oxidizes pyruvate to acetyl-CoA (AcCoA) leading to citrate synthesis or ketone-body formation. In the latter case pyruvate is converted to oxaloacetate by the action of pyruvate carboxylase (EC 6.4.1.1). Oxaloacetate may enter the gluconeogenic pathway by its conversion into phosphoenolpyruvate. Alternatively, it may depress ketogenesis by diverting AcCoA towards the synthesis of citrate which is considered to be a lipogenic precursor [I].

Accumulation of pyruvate by isolated rat liver mitochondria

1. Various methods to measure the rate of accumulation of [3-14C]pyruvate in the sucrose-impermeable space of isolated rat liver mitochondria are tested and compared with respect to their ability to distinguish between carrier-linked pyruvate transport and non-carrier-linked processes (adsorption and diffusion). 2. Evidence is presented that the cinnamic acid derivatives commonly used as specific inhibitors of the pyruvate carrier (i) do not completely abolish all carrier-mediated pyruvate transport; (ii) inhibit pyruvate adsorption, and (iii) at higher concentrations lead to a removal of previously accumulated pyruvate from the mitochondria. It is concluded that procedures which avoid the use of transport inhibitors allow more reliable estimates of carrier-linked pyruvate transport. 3. It is proposed to measure pyruvate adsorption as the accumulation of pyruvate in the presence of an uncoupler. Using this procedure, it could be shown that, with 1 mM pyruvate, adsorption represents only a small part of the total pyruvate accumulation, the main part being carrier-linked transport driven by the pH gradient across the mitochondrial inner membrane.

Acute effects of interleukin 1α and 6 on intermediary metabolism in freshly isolated rat hepatocytes

Using hepatocytes in suspension, freshly isolated from adult male fed rats, we studied the acute influence of recombinant human interleukins 1 alpha, 2 and 6 on glycogen and fatty acid metabolism. By far the largest effects were observed with interleukin-1 alpha: short incubations (up to 60 min) sufficed to depress glycogen synthesis in a dose-dependent manner, while the rates of glycogenolysis and glycolysis were increased as indicated by the release of glucose and lactate. Interleukin-6 acted similarly, though being much less effective on a molar basis, whereas interleukin-2 only caused a small increase in lactate production. In hepatocytes from 24h-starved rats interleukin-1 alpha caused a minor stimulation of gluconeogenesis. Although neither fatty acid synthesis nor oxidation of fatty acids in quiescent hepatocytes from fed rats was significantly affected by interleukins, interleukin-1 alpha was able to cause appreciable inhibition of fatty acid synthesis in hepatocytes from regenerating liver (isolated 22h after partial hepatectomy). It is concluded (i) that interleukins, in particular interleukin-1 alpha, acutely promote hepatic glucose release, and (ii) that transition of adult hepatocytes from a quiescent into a proliferatory state allows the occurrence of rapid effects of interleukin-1 alpha on fatty acid metabolism.

KETOGENESIS IN RAT-LIVER MITOCHONDRIA: STIMULATION BY PALMITYL-COENZYME A

It is well-known that the movement of adenine nucleotides (AdN) across the inner mitochondrial membrane is markedly decreased both by unsaturated [l41 and by saturated [4-61 long-chain fatty acids. A similar effect is displayed by palmityl-CoA as demonstrated recently with isolated mitochondria of rat heart [7] and rat liver [5,6,8-lo]. In fact, long chain acyl-CoA esters are much stronger inhibitors of mitochondrial AdN translocation than the corresponding free fatty acids and carnitine esters [6]. Therefore, long-chain acyl-CoA compounds such as palmitylCoA are the more likely candidates for the regulation of cell metabolism at the level of mitochondrial AdN transport. For a better appreciation of the possible physiological role of this inhibitory effect of palmityl-CoA, some of its metabolic implications in isolated rat-liver mitochondria were studied. In the present report the influence of small amounts of extramitochondrial palmityl-CoA on palmitate oxidation and concomitant ketogenesis is shown. Our results clearly indicate that the inhibition by palmityl-CoA of ADP uptake in the mitochondria is reflected in increased 3-hydroxybutyrate/acetoacetate (HB/Acac) ratios and in preferential conversion of acetyl-CoA (AcCoA) into ketone bodies. In a following publication [ 1 l] the stimulation of pyruvate carboxylation by added palmityl-CoA will be discussed.

Isozymic forms of protein kinase C in regenerating rat liver

The expression of multiple forms of protein kinase C (PK‐C) was studied in regenerating rat liver using hydroxyapatite column chromatography. Two forms of the enzyme were found in the cytosolic as well as membrane fraction of livers from partially hepatectomized rats. The kinetic variation in the activation of these two liver isozymes by fatty acids, phosphatidylserine and diacylglycerol was similar to that reported for the PK‐C subspecies from rat brain, designated types II and III. Intracellular redistribution of PK‐C caused by phorbol 12‐myristate 13‐acetate (PMA) was concentration‐dependent and was due to translocation of isozyme III, because type II was insensitive to 5 × 10−8 M PMA. The activity ratio of the two isozymes in either the particulate or cytosolic fraction was the same at 22 h as compared to 4 h after partial hepatectomy.

Levels of cyclic 3'-5' -adenosine monophosphate (cAMP) in maintenance cultures of rat hepatocytes in response to insulin and glucagon.

Under conditions of short-term stimulation of lipogenesis by insulin in maintenance cultures of hepatocytes from starved rats, basal levels of cyclic 3′-5′-adenosine monophosphate (cAMP) are invariant. Glucagon signals increased levels of cAMP and severely diminishes lipogenesis. Insulin partially counteracts both glucagon effects.

Effects of sodium 2-[5-(4-chlorophenyl)pentyl]-oxirane-2-carboxylate (POCA) on intermediary metabolism in isolated rat-liver cells

In hepatocytes isolated from meal-fed rats, sodium 2-[5-(4-chlorophenyl)pentyl]oxirane-2-carboxylate (POCA) decreased the rate of lipogenesis measured as incorporation of 3H from 3H2O into glycerolipids and cholesterol. Moreover, POCA inhibited the oxidation of added oleate, whereas oleate esterification was stimulated. In hepatocytes from 24-hr-starved rats, inhibition of gluconeogenesis by POCA was observed only with gluconeogenic precursors which require pyruvate carboxylation. This inhibition was secondary to impaired oxidation of long-chain fatty acids by POCA. It is concluded that, in addition to its inhibition of long-chain fatty acid oxidation, POCA interferes with de novo synthesis of cholesterol and fatty acids. On the other hand, neither fatty acid esterification nor the conversion of oxaloacetate into glucose are affected by POCA.

Differential short-term effects of growth factors on fatty acid synthesis in isolated rat-liver cells

Hepatocytes in suspension, freshly isolated from meal-fed rats, were used to study the acute influence of growth factors on the rate of de novo fatty acid synthesis. Nerve growth factor (2.5 S) and epidermal growth factor caused a substantial increase in the rate of fatty acid synthesis, whereas fibroblast growth factor was inhibitory. Little effect was observed with nerve growth factor (7 S), bombesin or substance P. Transferrin did not affect hepatic fatty acid synthesis. The results are discussed in relation to the effects of insulin and tumor-promoting phorbol esters.