The Regulation of Hepatic 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase

Easom, Richard A (1986) The Regulation of Hepatic 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase. PhD thesis, University of Glasgow.

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The aim of the work presented in this thesis was to assess the physiological significance and role of reversible phosphorylation-dephosphorylation in the short-term regulation of the activity of hepatic HMG-CoA reductase in vivo. Such a mechanism has been well characterized for this enzyme in vitro (see Chapter 1) but, until the present work no demonstration has been made of the influence of this system on the regulation of the enzyme in vivo in response to normal physiological stimuli (see Chapter 1 and Chapter 3). It seemed unlikely that such a complex mechanism of covalent modification would go unused in vivo. The previous work on whole animals was, therefore, reassessed. Particular care was taken in the sampling and homogenization of the tissue to preserve the phosphorylation status of the enzyme that exists in the tissue at the moment of sampling. Chapter 3 describes a technique, the cold-clamping technique, designed especially for this study. Not only did this technique meet the criterion for the rapid processing of liver tissue (see above) but it also allowed the physical removal of a HMG-CoA lyase activity (which interferes with the assay of HMG-CoA reductase) in whole mitochondria by differential centrifugation of the homogenates. The suitability of this technique for the study of short-term changes in HMG-CoA reductase activity was validated by directly comparing its efficiency in preserving the phosphorylation state of pyruvate kinase, previously established to undergo rapid interconversion between active and inactive forms, with the widely used freeze-clamping technique and the conventional method of mincing tissue prior to homogenization. Chapter 4 describes the use of the cold-clamping technique to quantitate changes in the fraction of the enzyme in the active, dephosphorylated form in a number of physiological conditions characterized by acute hormonal and/or nutritional changes. It was demonstrated that this parameter of HMG-CoA reductase showed a marked diurnal rhythm; its value was rapidly reduced under conditions of food deprivation for periods ranging from 4 to 24h. It also varied widely in rats during the transition between the gravid and lactating states. These changes appeared to occur independently of large changes in the total enzyme activity, (an indication of the amount of enzyme protein) suggesting that each parameter was controlled either by different hormones by a differential response to the same hormonal or nutritional factor. Significantly, the change in the phosphorylation status of the enzyme in each condition appeared to be the primary mechanism through which the expressed activity (the closest estimate of the actual activity in vivo of HMG-CoA reductase was rapidly modulated in co-ordination with the immediate physiological significance of the animal (see Section 4.5). In the final two chapters experiments are described which were designed to assess the importance of hormonal (Chapter 5) and/or nutritional (Chapter 6) factors in the short-term regulation of HMG-CoA reductase by reversible phosphorylation in vivo. A significant observation from Chapter 4 indicated that the changes in the fraction of enzyme in the active form in each physiological condition (except lactation) closely reflected the expected changes in the plasma insulin concentration in the animal. In Chapter 5, this suggestion was supported by, for example, the acute and chronic changes observed in this parameter in animals treated with anit-insulin serum and in severely insulin-dependent diabetic animals respectively. Further investigation into the role of hormones having antagonistic effects to insulin on liver metabolism (e. g. glucagon and epinephrine) revealed that insulin had a dominant role in the regulation of the phosphorylation state of HMG-CoA reductase in vivo. The observations are discussed in the light of the response of this parameter to different physiological conditions, in the light of the current knowledge of the regulation of cellular kinases and phosphatases for which HMG-CoA reductase enzyme is a substrate. In Chapter 6, the previously published effects of mevalonolactone on the phosphorylation state of HMG-CoA reductase both in vivo and in vitro were confirmed using the cold-clamping technique on the livers of animals intubated with mevalonolactone. Most significantly, however, this treatment produced a reversal of the dominant effect of insulin during the early hours of the dark period. The mechanism by which this could be achieved is discussed. This chapter also describes the apparently little effects of various dried milk fractions (with the exception of buttermilk) administered to animals by stomach intubation on the phosphorylation state of HMG-CoA reductase.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Biochemistry
Date of Award: 1986
Depositing User: Enlighten Team
Unique ID: glathesis:1986-77338
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 14 Jan 2020 11:53
Last Modified: 14 Jan 2020 11:53

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