Takhar, Sarvjinder; (1992) The regulation of mammalian lipogenesis via reversible phosphorylation by the camp-dependent and amp-activated protein kinases. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Lipid biosynthesis in the mammal is stringently regulated according to nutritional status. The rate limiting step in fatty acid synthesis is believed to be that catalysed by acetyl-CoA carboxylase (ACC). The activity of ACC is regulated both allosterically and by reversible phosphorylation. The work described in this thesis was conducted to ascertain which protein kinases are responsible for the in vivo phosphorylation and inactivation of ACC in the major lipogenic tissues i.e. lactating mammary gland and liver. Glucagon-mediated inhibition of ACC in hepatocytes, and phosphorylation and inactivation of purified ACC in vitro by purified cAMP-dependent protein kinase (cAMP-PK) from bovine heart suggested cAMP-PK as a good candidate for the physiological ACC kinase. However, intensive study of the catalytic subunit of cAMP-PK from mammary gland showed that it is probably an isozymic form of cAMP-PK characterised by poor stability and different substrate specificity to its cardiac counterpart, in particular a low affinity for ACC. Furthermore it appears that there is present in lactating mammary gland a factor that specifically inactivates the free catalytic subunit of cAMP-PK which may also be a reason for its lack of effect on ACC in vivo. Under physiological conditions eg over time courses of starvation, the inactivation of ACC in lactating rat mammary gland or rat liver more closely correlated with activation of AMP-activated protein kinase (AMP-PK) than cAMP-PK, This provided further evidence that AMP-PK and not cAMP-PK is the physiological ACC kinase. This work also suggested that the primary control point in the lactating mammary gland is pyruvate dehydrogenase (PDH) and its associated kinase, but in the liver ACC is the primary control point. AMP-PK itself is regulated by reversible phosphorylation, being phosphorylated and activated by a separate kinase kinase activity. A partial purification and characterisation of the AMP-PK kinase from mammary gland is described, together with its stimulation by fatty acyl-CoA. The final products of mammalian lipid biosynthesis are triglycerides and cholesteryl ester, these are assembled together to form very low density lipoprotein (VLDL) in the liver and are then secreted from the liver into the blood stream. The major structural protein of VLDL is apolipoprotein B-100 (apo B). Apo B is known to be a phosphoprotein in vivo. This work describes the in vitro phosphorylation of apo B by cAMP-PK and AMP-PK and identifies the sites of phosphorylation. It also reports the partial characterisation of a possibly novel apo B kinase.

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