Top–down control analysis (TDCA) is a useful tool for quantifying constraints on metabolic pathways that might be overcome by biotechnological approaches. Previous studies on lipid accumulation in oilseed rape have suggested that diacylglycerol acyltransferase (DGAT), which catalyses the final step in seed oil biosynthesis, might be an effective target for enhancing seed oil content. Here, increased seed oil content, increased DGAT activity, and reduced substrate:product ratio are demonstrated, as well as reduced flux control by complex lipid assembly, as determined by TDCA in Brassica napus (canola) lines which overexpress the gene encoding type-1 DGAT. Lines overexpressing DGAT1 also exhibited considerably enhanced seed oil content under drought conditions. These results support the use of TDCA in guiding the rational selection of molecular targets for oilseed modification. The most effective lines had a seed oil increase of 14%. Moreover, overexpression of DGAT1 under drought conditions reduced this environmental penalty on seed oil content.

Top-Down (Metabolic) Control Analysis (TDCA) was used to examine, quantitatively, lipid biosynthesis in tissue cultures from two commercially important oil crops, olive (Olea europaea L.) and oil palm (Elaeis guineensis Jacq.). A conceptually simplified system was defined comprising two blocks of reactions: fatty acid synthesis (Block A) and lipid assembly (Block B), which produced and consumed, respectively, a common and unique system intermediate, cytosolic acyl-CoA. We manipulated the steady-state levels of the system intermediate by adding exogenous oleic acid and, using two independent assays, measured the effect of the addition on the system fluxes (J(A) and J(B)). These were the rate of incorporation of radioactivity: (i) through Block A from [1-(14)C]acetate into fatty acids and (ii) via Block B from [U-(14)C]glycerol into complex lipids respectively. The data showed that fatty acid formation (Block A) exerted higher control than lipid assembly (Block B) in both tissues with the following group flux control coefficients (C):(i) Oil palm: *C(J(TL))(BlkA)=0.64+/-0.05 and *C(J(TL))(BlkB)=0.36+/-0.05(ii) Olive: *C(J(TL))(BlkA)=0.57+/-0.10 and *C(J(TL))(BlkB)=0.43+/-0.10where *C indicates the group flux control coefficient over the lipid biosynthesis flux (J(TL)) and the subscripts BlkA and BlkB refer to defined blocks of the system, Block A and Block B. Nevertheless, because both parts of the lipid biosynthetic pathway exert significant flux control, we suggest strongly that manipulation of single enzyme steps will not affect product yield appreciably. The present study represents the first use of TDCA to examine the overall lipid biosynthetic pathway in any tissue, and its findings are of immediate academic and economic relevance to the yield and nutritional quality of oil crops.

The Flux Control Coefficients of mitochondrial outer membrane carnitine palmitoyltransferase (CPT I) with respect to the overall rates of beta-oxidation, ketogenesis and tricarboxylic acid cycle activity were measured in hepatocytes isolated from rats in different metabolic states (fed, 24 h-starved, starved-refed and starved/insulin-treated). These conditions were chosen because there is controversy as to whether, when significant control ceases to be exerted by CPT I over the rate of fatty oxidation [Moir and Zammit (1994) Trends Biochem. Sci. 19, 313-317], this is transferred to one or more steps proximal to acylcarnitine synthesis (e.g. decreased delivery of fatty acids to the liver) or to the reaction catalysed by mitochondrial 3-hydroxy-3-methyl-glutaryl-CoA synthase [Hegardt (1995) Biochem. Soc. Trans. 23, 486-490]. Therefore isolated hepatocytes were used in the present study to exclude the involvement of changes in the rate of delivery of non-esterified fatty acids (NEFA) to the liver, such as occur in vivo, and to ascertain whether, under conditions of constant supply of NEFA, CPT I retains control over the relevant fluxes of fatty acid oxidation to ketones and carbon dioxide, or whether control is transferred to another (intrahepatocytic) site. The results clearly show that the Flux Control Coefficients of CPT I with respect to overall beta-oxidation and ketogenesis are very high under all conditions investigated, indicating that control is not lost to another intrahepatic site during the metabolic transitions studied. The control of CPT I over tricarboxylic acid cycle activity was always very low. The significance of these findings for the integration of fatty acid and carbohydrate metabolism in the liver is discussed.

Summary• Metabolic control analysis allows the study of metabolic regulation. We applied both singleand double-manipulation top-down control analysis to examine the control of lipid accumulation in developing oilseed rape (Brassica napus) embryos.• The biosynthetic pathway was conceptually divided into two blocks of reactions (fatty acid biosynthesis (Block A), lipid assembly (Block B)) connected by a single system intermediate, the acyl-coenzyme A (acyl-CoA) pool. Single manipulation used exogenous oleate. Triclosan was used to inhibit specifically Block A, whereas diazepam selectively manipulated flux through Block B.• Exogenous oleate inhibited the radiolabelling of fatty acids from [1-14 C]acetate, but stimulated that from [U-14 C]glycerol into acyl lipids. The calculation of group flux control coefficients showed that c. 70% of the metabolic control was in the lipid assembly block of reactions. Monte Carlo simulations gave an estimation of the error of the resulting group flux control coefficients as 0.27 ± 0.06 for Block A and 0.73 ± 0.06 for Block B.• The two methods of control analysis gave very similar results and showed that Block B reactions were more important under our conditions. This contrasts notably with data from oil palm or olive fruit cultures and is important for efforts to increase oilseed rape lipid yields.

1. 3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase (EC 4.1.3.5) in extracts of rat liver mitochondria can be inactivated by succinyl-CoA and activated by incubation in a medium designed to cause desuccinylation ('desuccinylation medium'). 2. The enzyme is less active in extracts of whole liver from control rats than from rats treated with glucagon or mannoheptulose. Incubation in desuccinylation medium raises the activity in extracts from control rats to the same value as treated rats, suggesting that the extent of succinylation in vivo is greater in controls than in hormone-treated animals. 3. This result is also obtained in liver homogenates and in isolated mitochondria. 4. Increasing the succinyl-CoA content of mitochondria to the same high level lowers the enzyme activity to the same value in mitochondria isolated from control or treated rats. In each case subsequent incubation of the lysates in desuccinylation medium raises the enzyme activity by the same extent. 5. Measurement of the incorporation of radiolabel from 2-o~o[S-'~C]glutarate into protein is consistent with the proposal that all these changes in activity in isolated mitochondria may be explained by changes in the extent of succinylation of the enzyme. 6. From these data and our earlier work we conclude that, in vivo, mitochondrial HMG-CoA synthase in fed rats is normally substantially succinylated (about 40%) and inactivated, and that glucagon increases the activity of HMG-CoA synthase by lowering the concentration of succinyl-CoA and thus decreasing the extent of succinylation of the enzyme (to less than 30%). This may be an important control mechanism in ketogenesis.The control of ketogenesis in liver is not fully understood [I -31 but it is clear that the production of ketone bodies can be stimulated by treatment of animals with glucagon [4-91. Although there is good evidence that some control of ketogenic flux is exerted at carnitine palmitoyltransferase I [3, 9 -121, there is also strong evidence that under certain conditions control shifts to an unidentified intramitochondrial control site [13-201. On the basis of experiments in vitro with mitochondrial HMG-CoA synthase purified from ox liver, Lowe and Tubbs [21] proposed that glucagon might increase ketogenic flux through the HMG-CoA pathway by lowering the succinylCoA concentration and therefore the succinylation state of HMG-CoA synthase.In support of this hypothesis we have observed increased HMG-CoA synthase activity and decreased intramitochondrial succinyl-CoA levels in extracts prepared from rapidly frozen livers of rats after short-term treatment with glucagon or mannoheptulose. These effects persisted in isolated mitochondria, validating their use as a model system in which to examine the effects of hormones on HMG-CoA synthase. We also showed that succinyl-CoA inhibited HMG-CoA synthase activity in isolated mitochondria and that the differences in the activity of the enzyme in mitochondria isolated from glucagon-treated or mannoheptulose-treated rats and shamCorrespondence ...