Tey, Kim Kuan; (1996) Regulation of fatty acid synthesis pathway in the lactating rat mammary gland. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The lactating rat mammary gland is highly active in synthesising fatty acids and isolated mammary acini were used to investigate the regulation and interdependence of specific regulatory steps in the pathway from glucose to fatty acids. Various putative effectors of lipogenesis were used to investigate the control and relative importance of these specific regulatory steps. Acetoacetate, a potential physiological signal in starvation inhibited fatty acid synthesis (38%) but not acetyl-CoA carboxylase (ACC). Surprisingly, ACC was activated and this survived purification (Vmax increased by 100%). ATP-citrate lyase (ATP-CL) and pyruvate dehydrogenase (PDH) were inhibited by 18% and 83%, respectively. However, neither of these are likely to account for the lipogenic inhibition, given the increase in citrate concentration caused by acetoacetate. Glucose uptake was reduced by 54% in the presence of acetoacetate, but this was not the result of a decrease in the rate of glucose transport which was unaffected. The data suggest that reduced glucose supply to the cells is the major factor in lipogenic inhibition, possibly via the consequent reduced production of NADPH for fatty acid synthesis, or glycerol-3-phosphate for esterification. Inhibition of glycolysis probably occurs as a result of phosphofructo-1-kinase (PFK-1) inhibition by the elevated citrate levels in combination with PDH inactivation. Investigation of the profile of protein phosphatase activity in mammary gland showed the presence of okadaic acid-sensitive PP1 and PP2A. Okadaic acid is a potent cell-permeable inhibitor of PP1 and PP2A. It inhibited fatty acid synthesis (63%) without affecting the rate of glucose uptake by rat mammary acini. While GLUT 1 (the predominant glucose transporter in mammary acini) like GLUT 4 (the insulin stimulated glucose transporter) can be regulated by translocation to the plasma membrane, these studies confirm that phosphorylation is unlikely to be involved in this regulation. ACC was phosphorylated and inactivated (Vmax decreased by 70%) in response to okadaic acid. This is an expected result of PP2A inhibition but a transient activation of AMP-PK was also observed. Time course studies showed that PDH inhibition in response to okadaic acid was a consequence of the inhibition of ACC, presumably via an increased acetyl CoA/CoA ratio that would activate PDH kinase. The polyunsaturated fatty acid, eicosapentanoic acid (EPA), inhibited fatty acid synthesis in mammary acini in a dose-dependent manner. Maximal inhibition was achieved at 1mM EPA and at this concentration both PDH and ACC were inhibited. Linolenic and linoleic acids produced similar inhibitory effects with the degree of inhibition depending on the degree of unsaturation of the fatty acid. Clofibrate and gemfibrozil are lipid-lowering drugs with structures analogous to those of fatty acids. These drugs also inhibited fatty acid synthesis in acini (33% and 84%, respectively) via PDH and ACC inactivation. In vivo studies with gemfibrozil indicated that doses of this agent known to produce peroxisomal proliferation produced similar short term inhibition of fatty acid synthesis (68%), and cholesterol synthesis (81%) in rat liver in vivo. Surprisingly, fatty acid synthesis in white adipose tissue was increased by 71%.

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