Diabetes research background
©
R.J.Walters Ph.D., 2003.
The Beta Cell - the epicenter of diabetes
As the Beta-cell is the source of insulin secretion, it is inevitably the focus of attention
in how changes of blood glucose are detected and transduced (converted) into changes in insulin release,
what scientists like to call stimulus-secretion coupling. For this to happen, we must know how glucose
enters the beta-cell, how it is converted (metabolised) into a messenger, or messengers, and how these
changes in messenger levels are detected and converted into the changes of electrical activity and the entry of calcium ions into the cell that
stimulate the release of insulin. We know that the glucose transporter GLUT-2 mediates the uptake of
glucose, and that most of it is converted into ATP by the glycolysis pathway. However, what remains unclear
is what receptors other than the KATP potassium channel sense changes in ATP levels, and which other glucose
(or alternative fuel) metabolites are involved.
There remains much controversy regarding the nature and distribution of
calcium channels in the pancreatic B-cell membrane. The understanding of their
distribution, regulation and voltage-dependence is of critical importance in
developing therapeutic strategies in the treatment of both type I and II
diabetes, as calcium has been reproducibly demonstrated to be the principal
effector in coupling changes in glucose concentration to changes in insulin
release. Whilst there may be other modulators and effectors governing insulin
release, the concentration of free calcium in the cell shows a linear
correlation with insulin release in isolated islets (Jonas et al. 1998, it
should however be noted that high affinity calcium indicators such as Fura II
significantly distort the temporal and kinetic nature of calcium transients as compared
with low affinity indicators such as Magnesium Green). Therefore the characterization of the
different populations of calcium channels underlying the complex kinetics of
calcium changes in the B-cell is of central importance to understanding and
controlling insulin release.
Current-clamp and perforated-patch clamp recordings were made
from cultured pancreatic B-cells to investigate the behavior and regulation of
novel calcium channels and also to study evidence for their direct modulation
by sulphonylureas. The modulation of membrane potential by D-glucose was
investigated under a range of conditions, and measurements of changes in free
calcium in response to D-glucose and elicited depolarizations were studied by calcium
fluorescence imaging.
Voltage-step and paired-pulse protocols, ion substitution experiments and
pharmacology were used to identify and discriminate between two kinetically
distinct calcium channel populations in the B-cell, both of which showed a
clear and reversible activation by elevations in the concentration of
D-glucose. Using pseudo-physiological solutions has allowed the observation of
the 'normal' kinetic and permeation properties of these two novel ion channel
populations, and their selectivity for calcium ions was deduced by current
polarity (K+ currents yield positive current deflections and Na+/Ca2+
currents inward deflections), reversal potential and by ion substitution
experiments.
Studies of their regulation by different metabolic substrates and inhibitors
and their voltage kinetics were pursued. The difference in findings with
earlier classical studies have been replicated and explained in terms of the
specific non-physiological parameters used in these investigations. It was found both in this laboratory
and subsequently that tetraethylammonium (TEA+) distorts the kinetics of
calcium currents, as does the introduction of calcium chelators such as BAPTA
and EGTA. Thus pseudophysiological
measurements with perforated-patch clamp ought also to be performed in parallel
to provide a ‘control’ for calcium current measurements.
Of special interest is the activation of G-type currents, but not L-type channels, by the ketogenic amino acids L-Leucine and L-glutamine,
and the observation that glucose-mimetic (imitating) sulphonylureas activate L-type, but not G-type channels, directly in the absence of glucose.
This suggests that L and G-type calcium channels are regulated very differently. L-type calcium channels are switched on directly by imitating the
action of glucose at the sulphonylurea receptor, whilst KATP channels are switched off by the same mechanism. Similarly the fact the ketogenic
amino acids activate the G-type channel alone, suggests that there is another metabolite other than ATP which regulates insulin secretion.
The properties of the G- and L-type calcium channels allude to distinct
roles in the regulation of calcium homeostasis in the B-cell, but do not
necessarily fully explain the dynamics of calcium entry and insulin release.
However these more physiological studies help to bridge the divide between what
is understood from studies in whole islets and apparently contradictory
findings in studies of calcium currents in single isolated B-cells, as no beta
cell was ever intended to be an island.
Insulin receptors - the other side of the equation
Much of the author's diabetes research has focused on how insulin signals are turned into cellular events within the target cells of circulating insulin.
This is rather a diffuse statement, as in fact most cells possess insulin receptors. Other than its action as a regulator of
blood glucose levels, insulin is also a potent growth factor (much abused by bodybuilders) and regulates diverse cellular processes, including growth and division.
Studies of the myeloid-derived cell line U937 (a cell line of white blood cell lineage which has been used as a model for monocytes and previously neutrophils),
shows that insulin stimulates polmerization of the cell's actin cytoskeleton, an action which might affect the migration of white blood cells as they adhere and leave the circulation en route to peripheral tissues.
Further, we found that insulin exerts many effects upon PC12 cells, a cell line which can, under the right conditions, be induced to behave as either nerve cells (releasing processes and releasing dopamine and/or noradrenaline) or adrenal chromaffin cells (releasing adrenaline) in culture.
Surprisingly insulin affected virtually every process studied in the PC12 cell. Insulin stimulates the outgrowth of processes (neurites) from PC12 cells, stimulates the aggregation of actin into
focal clusters (islands on the cell surface) which coincide in space with clustering of GABA-A receptors. Further, insulin alters the level of expression of GABA-A receptors themselves, and
inhibits the expression of the enzymes responsible for the production of noradrenaline (DBH) and adrenaline (PNMT), but not that of the rate-limiting enzyme for the production of Dopamine (Tyrosine Hydroxylase).
Thus behavior itself is regulated by insulin, as a decrease in adrenaline and noradrenaline levels would be expected to decrease alertness, vigilance and aggression.
Therefore insulin is far more that just a regulator of blood sugar levels, as it controls the growth, form and activity status of virtually every type of cell in the body, and thus serves
as a key determinant of the set-point of physiological activity, which inevitably revolves around cycles of fasting and eating, activity and inactivity, wakefulness and sleep.
Dysfunctions of the insulin signaling system characteristic of diabetes likely disrupt many more processes than just the levels of resting glucose in the blood.
