A study of the regulation of hepatic microsomal glycerol phosphate acyltransferase (GPAT) : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University
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1986
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Massey University
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Abstract
Experiments described in this thesis were conducted to examine the
possibility that hepatic microsomal GPAT activity in rats is regulated
by insulin.
Hepatic microsomal fractions were prepared by a procedure based on
published methods and it was established by assay of cytochrome
oxidase, monoamine oxidase and NADPH cytochrome Creductase that there
was less than 11% mitochondrial impurity. A butanol extraction of GPAT
assays was adopted to separate the butanol-soluble [14cJ-lipid products
from the unreacted aqueous-soluble [ 1 4C ) -glycerol 3-phosphate
substrate . Methods were developed to simplify the determination of
[14 c J-radioactivity. The kinetics of the response of the assay system
to changes in the concentrations of glycerol 3-phosphate and
palmitoyl-CoA were similar to those published for the microsomal GPAT.
The products of the assay were identified as phosphatidic acid and
lysophosphatidic acid by their chromatographic properties before and
after hydrolysis with chicken liver phosphatidate phosphohydrolase.
These products are consistent with literature reports.
Male Sprague-Dawley rats were treated with insulin (4i.u./kg body
weight) orsaline, and extracts of hind-limb muscle were prepared and
fractionated on Sephadex G-25 in 5 0 mM formic acid. Fractions which
eluted subsequent to the void volume were assayed with hepatic
microsomal GPAT and the effect of insulin-treatment fractions were
compared with the effect of saline-treatment fractions.
Fractions containing material of approximately 3000 and 1000 daltons
molecular weight enhanced GPAT activity in an insulin-dependent manner
by 0.46 and 0.64 nmol/min/mg of microsomal protein, respectively (both
P<0.01), compared to the effect of the saline controls. Control rates
were approximately 3.5nmol/min/mg of microsomal protein. It was
calculated that these insulin-dependent increases in hepatic microsomal
GPAT activity would be sufficient to account for the difference between
the estimated hepatic triacylglycerol production of fed and fasted
rats. Furthermore , published studies suggest that insulin-dependent
changes in activities of enzymes, demonstrated with in vitro systems
utilising low molecular weight fractions from rat muscle, may parallel
sensitivity of the same enzymes to insulin in vivo. The low molecular
weight stimulator or stimulators of hepatic microsomal GPAT have an
apparent molecular weight within the range 1000-3000 daltons, appear to
be heat and acid stable, are soluble in aqueous solution, have very low
absorbance at 220nm (or a very high specific activity) and may be
sensitive to oxygen. These properties suggest that the low molecular
weight stimulator or stimulators of hepatic microsomal GPAT activity
may be related to the putative insulin mediator substance (IMS).
In initial experiments, where rats were heparinised prior to
treatment with insulin or saline, it was observed that some fractions
were able to stimulate hepatic microsomal GPAT activity in an
insulin-independent manner. Experiments to resolve this suggested that
the treatment of rats with heparin alone led to the presence of low
molecular weight material, in the fractions of muscle extracts, with
the potential to enhance GPAT activity. It was found that low
molecular weight fractions of the saline treatment muscle extracts did
not enhance GPAT activity. This supported the suggestion that heparin
was responsible for the ability of low molecular weight fractions of
muscle extracts to stimulate GPAT activity in an insulin-independent
manner.
Experiments were also conducted in which impure hepatic plasma
membranes were treated with insulin (20-1000 units/ml). However, when
hepatic microsomal GPAT was assayed with material from these
incubations a stimulator of GPAT was not detected.
The results of experiments presented in this thesis provide
further evidence in favour of the hypothesis that hepatic microsomal
GPAT activity can be modified by insulin and may contribute to the
overall regulation of glycerolipid biosynthesis in liver.
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Keywords
Glycerol phosphate acyltransferase, Rats, Insulin, Heparin