The work to be described was undertaken in
order to substantiate and extend the work of
Fisher and Zachariah (1961) on the action of
insulin on the penetration of non -metabolised
pentoses into the cells of the perfused rat heart.
These workers found that sugar uptake by this preparation conformed best to kinetics based on the
carrier transport hypothesis of ',Widdas (1954),
whereby the sugar is assumed to be transported
across the cell membrane in combination with a
carrier molecule and released into the cell.
Fisher and Zachariah used a known constant
concentration of either xylose or arabinose in the
medium perfusing the rat heart, and estimated
values of f after different periods (t) of perfusion. They were then able to determine the
values of K and V for each sugar which gave closest conformity of the results to the carrier hypothesis.
They found that in the absence of insulin the
values of K for the sugars were very small indicating that the carrier molecule had a very high
apparent affinity for the sugars. When a low, sub - maximal concentration of insulin was added to the
perfusate, the values of K were increased indicating that insulin diminished the apparent affinities
of the carrier for the sugars, but the values of V
One might expect intuitively that insulin
would accelerate sugar transport by increasing the
affinity of the carrier for a sugar but Fisher and
Zachariah were able to show, on the basis of the
carrier model, how sugar transport would be
accelerated when the apparent affinity of the
carrier for the sugar was decreased and it was
assumed that this occurred as a result of a combination of insulin with the carrier. They proposed a hypothesis of insulin action based on
their findings and accounted for the diminution in
V brought about by insulin by assuming that in the
presence of insulin sugar transport would be mediated almost entirely by that fraction of the
carrier which had been modified by combination
with insulin, so that the total amount of carrier
(C) involved in effective sugar transport would be
smaller than that involved in transport when no
insulin was present.
There were several gaps in the evidence produced by fisher and Zachariah in support of the
carrier mechanism of sugar transport and of their
suggestion as to how insulin might effect this
(i) It is a property of equation (1) that the
higher the sugar concentration outside the cells,
so the lower should be the value of f at any
given time. Fisher and Zachariah used only one
sugar concentration throughout their experiments,
so that this test of the equation remains to be
(ii) These workers used only one insulin concentration in their experiments. Their hypothesis predicts that if the insulin concentration is
increased above the submaximal concentration which
they used, then the value of K for any sugar should
not alter further, but V should increase because
the fraction of total carrier involved in effective transport (i.e. that fraction modified by
insulin) should increase. The limiting value of
V, at the insulin concentration having the greatest effect on sugar transport, should therefore be
the same as the value found in the absence of
insulin, since all the carrier should once again
be involved in the transport process.
In addition to these gaps in the evidence,
disquieting feature of the work of Fisher and
Zachariah was the variability of the sugar- uptake
results, and there seemed to be two main reasons
(i) The analytical techniques involved in the
sugar uptake measurements provided a source of
variation which it was felt might be better controlled by using automatic estimation techniques.
(ii) Another source of variation lay in the
response of the animals used in these experiments
to variations in environmental factors from day to
day, (Young, 1960). This should be eliminated by
keeping the animals under conditions of constant
temperature and by regulating the poriod of daily
illumination by artificial means.
Two other major advances have been made in
improving the techniques involved in this work.
Firstly, the perfusion apparatus has been modified
so that continuous filtration of the medium per -
fusing the heart is achieved by means of a hardened paper filter lather than the sintered glass
filter used by Fisher and Zachariah. With the
paper filter present it has been found that hearts
can be maintained on a modified Krebs bicarbonate
medium containing pyruvate to act as nutrient
without including in this medium the plasma protein which Fisher and Zachariah had found to be
necessary to sustain the hearts in a state of
normal contractility in their apparatus. Thus
permeability is studied in the present work in the
absence of the complicating influence of a plasma
protein preparation of undetermined composition.
Secondly, the technique of estimating K and V from
the experimental data has been improved,
In the work to be described, therefore, the
work of Fisher and Zachariah is repeated using
considerably improved techniques. In addition,
the predictions which were made on the basis of
their hypothesis are tested thoroughly. The
uptakes of xylose and arabinose by the perfused
heart have been studied over a range of perfusate
sugar concentrations both in the presence and
absence of different insulin concentrations.
The results confirm the prediction that the
transport process conforms to the carrier mechanism at all the sugar concentrations studied.
The findings of Fisher and Zachariah that the
values of K for the pentoses are very small in the
absence of insulin, and that the values are
increased by insulin are also confirmed. AS the
insulin concentration of the perfusate is increased,
the K values do not alter significantly and this
is again in agreement with the prediction.
However, as the insulin concentration is increased,
the values of V for the sugars increase in approximate proportion, so that at high insulin concentrations they greatly exceed the value which
is predicted to be the limiting one on the basis
of the original hypothesis,
At first sight these findings suggest that
insulin has two actions of permeability, : --
(i) an all -or -none action in increasing K, and
(ii) a graded action in increasing V.
However, both K and V are functions of the rate
constant k3 of sugar -carrier complex movement
through the membrane, (see equation (1)) and it
will be shown that the two effects can be accounted
for completely in terms of the effect of insulin
on this rate constant.
The resultant hypothesis of insulin action
can be described in simple physico -chemical terms
as an effect of insulin on the tendency of the
mobile carrier molecule to associate with fixed
elements in the cell membrane.