SHORT TERM REGULATION Allosteric Effectors

When rats are fasted for less than 24 hours or subjected to acute insulin deficiency the decrease in the activity of ACC is greater than explained by enzyme turnover. The activity of ACC can be altered in vitro allosterically by citrate (Martin and Vagelos 1962, Moss and Lane 1971) and fatty acyl-CoA thioesters (Bortz and Lynen 1963; Lunzer et al 1977; Ogiwara et al 1978) which activate and inhibit the enzyme respectively. In vitro inhibition of ACC by fatty acyl-CoA esters is competitive with citrate (Goodridge 1972; Halestrap and Denton 1974;

Ogiwara et al 1978) and acetyl-CoA (Bortz and Lynen 1963) . This evidence would suggest a plausible mechanism for the regulation of ACC is the forward activation by citrate (the immediate precursor for acetyl-CoA) antagonised by end product inhibition by fatty acyl-CoA thioesters.

In vivo there is some evidence to support this. The concentration of citrate required for the half maximal activation (Ka citrate) of ACC from mammary gland is approximately 2 mM (Munday and Hardie 1884, 1986) and the intracellular concentration varies between 0.2 to 0.5 mM (Williamson et al 1975, Robinson and Williamson 1977b) making it a potential physiological regulator. However, there is no conclusive evidence showing a close correlation between fatty acid synthesis and citrate levels. Nishikori et al (1973) reported a rapid rise in citrate and fatty acid synthesis in the liver of starved and re-fed rats. To contrast , in rat epididymal fat pads stimulation of fatty acid synthesis by insulin was associated with an unchanged or decreased tissue concentration of citrate whereas adrenaline caused an increase in citrate concentrations along with a decrease in fatty acid synthesis. (Denton and Halperin 1968, Saggerson and Greenbaum 1970, Halestrap and Denton 1974). By using fluoroacetate which is metabolised to fluorocitrate a potent inhibitor of aconitase it is possible to raise citrate levels ten-fold. When this was done using epididymal fat pads no marked

increase in the initial activity of ACC was observed (Brownsey et al 1977).

An inverse relationship between the rate of fatty acid synthesis and intracellular levels of fatty acyl-CoA esters has been demonstrated in rat epididymal fat pads treated with insulin (Denton and Halperin 1968; Saggerson and Greenbaum 1970) also in the liver of starved rats refed a fat free diet (Nishikori et al 1973). Jacobs and Majerus (197 3) using skin fibroblasts found no such relationship. The role of fatty acyl-CoA esters in vivo will be extremely difficult to resolve largely because the free intracellular

levels are difficult to measure. They are modulated by fatty acid binding proteins (Lunzer et al 1977) and also compartmentalization within the cell. It is estimated that a total fatty acid concentration of 50 - 150 juM is reduced to a free concentration of 1.5 - 15 fiM. (Brownsey and Denton 1987, Lunzer et al 1977) but the Ki of palmitoyl-CoA for ACC has been reported as <10 nM (Ogiwara et al 1978, Nikawa et al 1979) . This would mean that free fatty acids are always at inhibitory levels, and thus lessens the likelihood that they are physiological mediators.

Several other potential regulators of ACC have been described. These include GTP and other guanine nucleotides (Witters et al 1981, Beuchler and Gibson 1984), polyphosphoinositides especially phosphatidyl inositol 4,5- bisphosphate (Heger and Peter 1977, Blyth and Kim 1982), an autocrine factor tentatively identified as an oligosaccharide (Witters et al 1988), small molecular weight substances released from liver plasma membranes by insulin binding (Saltiel et al 1983) and ADP-ribosylation (Witters and McDermott 1986). The physiological relevance of these potential regulators is still unclear.

ACC polymerises to form linear helices of up to 30 dimers in length (Ahmed et al 1978). Citrate induces this polymerisation in vitro and fatty acyl-CoA esters cause disaggregation. The filamentous form of ACC is more active than the protomeric dimer but activation of the ACC subunits precedes their polymerization in the presence of citrate (Beaty and Lane 1983). There is evidence that the polymerization of ACC occurs when it is activated irrespective of the effector agent. Thus ACC activation by Coenzyme-A (Yeh et al 1981) or limited trypsinolysis (Iritani et al 1969) both induce polymerisation. The possible importance of polymerisation in vivo has been considered by Borthwick et al (1987) who have reported that ACC from insulin treated tissue which has elevated ACC activity is more highly polymerised in the absence of citrate than that from tissue treated with the B-agonist

isoprenaline which inhibits lipogenesis. Similarly Ashcraft et al (1980) have reported that fat feeding or starvation, both of which decrease lipogenesis, cause a decrease in the amount of polymeric ACC in chicken liver. Thus indicating that the polymerisation of ACC may be of significance in v i v o . Tanake et al (1977) have reported that ACC is less susceptible to proteolysis in the presence of citrate when it is in the polymeric form.

Allred and co-workers have published work describing the regulation of ACC by the translocation of an inactive form of the enzyme from the mitochondrial outer membrane to the cytosol where upon the enzyme becomes active (Allred and Roman-Lopez 1988). The proportion of active cytosolic to inactive mitochondrial ACC is decreased by fasting and increased by refeeding (Allred et al 1985; Roman-Lopez et al 1989). These workers also show short term insulin deficiency increases the amount of inactive ACC associated with the mitochondria. (Roman-Lopez and Allred 1987) thus providing a mechanism for the decreased lipogenesis seen in diabetic animals It has also been shown that the increased ACC activity characteristic of genetically obese Zucker rats is not due to a greater amount of enzyme but is the result of a greater proportion of ACC being in the active cytosolic form (Allred et al 1989). All this work has been done in rat liver but a similar mechanism if it occurs in the mammary gland would allow rapid control of ACC activity. It must be noted however that Moir and Zammit (1990) have reported in direct contrast that the hepatic mitochondrial ACC pool does not replenish the cytosolic ACC upon refeeding of starved rats. There are no reports addressing possible interactions between enzyme translocation, and the allosteric and covalent modification mechanisms for controlling ACC activity.