The Role of Insulin and Glucagon in the Regulation of Hepatic Drug and Steroid Metabolism

Hussin, Abas Bin Haji (1988) The Role of Insulin and Glucagon in the Regulation of Hepatic Drug and Steroid Metabolism. PhD thesis, University of Glasgow.

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The object of this thesis is to ascertain the role of insulin and glucagon in the regulation of steroid metabolism in the rat liver. Most of the previous work performed to observe the effect of diabetes on steroid and drug metabolism was done on liver microsomes prepared from treated animals. As described above, it is difficult to ascribe the effect of diabetes on hepatic steroid metabolism to a single action of insulin or glucagon in an in-vivo study. The use of isolated hepatocytes is essential to examine the effect of one hormone alone. In order to achieve our objective, we had to develop a cell culture system which is hormone- and serum-free. We have developed and characterized four different types of culture medium containing animal serum, synthetic multihormone serum substitute (Ultroser G, LKB ) or bovine serum albumin only. When the liver cells were cultured in basic Ham's F-10 culture medium supplemented with foetal calf and horse serum or with Ultroser G, the steroid enzyme activities were reduced to less than 50 % of control on day 3 of culture in normal male rat hepatocytes. We discovered that the steroid enzyme activities were best maintained in Ham's F-10 culture medium supplemented with 0.1% bovine serum albumin only. The basal level of enzyme activities was maintained for at least 3 days in culture. We, therefore, have a hepatocyte culture method to assess the effects of insulin and glucagon on hepatic steroid metabolism in serum- and hormone-free, chemically defined medium. Only male rats were used throughout the project because previous study had shown that the effect of diabetes on hepatic steroid metabolism is only seen in the male rat (Skett, 1986). The substrate chosen for the investigation is androst-4-ene-3,17-dione because its metabolites are well defined and easily separated and the labelled and unlabelled substrate are easily available. The enzymes involved in its metabolism have been shown to be both cytochrome P-450-dependent and -independent and sex-specific. Moreover, insulin has been demonstrated to affect its metabolism in-vivo (Skett, 1986). The effect of insulin on androst-4-ene-3,17-dione metabolism in normal rat hepatocytes over the period studied was characterized by the presence of two peaks of increased activity at 1/2 and 24 hours. Importantly, these effects of insulin are clearly within the physiological range. The dose-response curves at 1/2 and 24 hour insulin preincubation suggest that these two peaks are probably generated by different mechanisms. Biochemical studies performed indicated that there is no correlation between the increase in enzyme activities by insulin at 1/2 hour and changes in cyclic AMP, cytochrome P-450 concentrations or phosphoinositide hydrolysis. It seems that insulin's effect on androst-4-ene-3,17-dione metabolism at 1/2 and 24 hour involves a phosphorylation reaction since the protein kinase inhibitor, K-252a, completely inhibited the effect of insulin. However, the exact molecular mechanism of action is yet to be determined. Our results did not show any selective changes in the male-specific and female-specific enzyme activities which is in contrast to the result found in-vivo (Skett, 1986). This is probably attributed to the continuous exposure of the parenchymal liver cells to many different hormones in-vivo, which is absent in our system. The data obtained suggest that insulin has a direct effect on hepatic steroid metabolism and the hormone, in-vitro, act as a general stimulator of the enzymes in the liver which metabolize androst-4-ene-3,17-dione. (Abstract shortened by ProQuest.).

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Pharmacology
Date of Award: 1988
Depositing User: Enlighten Team
Unique ID: glathesis:1988-77742
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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