Download Effects of insulin under normal and low glucose on retinal

Effects of Insulin Under Normal and Low Glucose on
Retinal Electrophysiology in the Perfused Cat Eye
Nicola hansel and Gilnter Niemeyer
Purpose. To investigate the short-term effects of fast-acting insulin on the electroretinogramb-wave, optic nerve response, standing potential, and flow rate in the arterially perfused cat
eye under normal conditions and during low glucose levels.
Methods. Enucleated cat eyes were perfused with a glucose- and insulin-free tissue culture
medium, to which glucose was applied at normal (5.5 mM) and reduced (2 and 1 mM)
concentrations. Photic stimulation was performed in the rod-matched intensity range before,
during, and after insulin application at postprandial (5 ng/ml) and at 10 and 20 X higher
concentrations.
Results. Insulin failed to affect retinal signals at normal glucose levels. However, insulin enhanced the low glucose-induced decrease in rod-driven b-wave amplitude (P < 0.05 at 2 mM;
P < 0.01 at 1 mM) without affecting the corresponding changes in the optic nerve response.
The standing potential increased by as much as 0.75 mV in response to insulin. The perfusate
flow rate was not altered by insulin.
Conclusions. Insulin was not required for normal retinal function as observed during 10 hours
of perfusion. The differential responsiveness to insulin under low glucose of the b-wave versus
the optic nerve response is thought to reflect suppression of glucose use by Muller (glial)
cells rather than neuromodulation, as the neuronal optic nerve response is unaffected. The
postulated insulin sensitivity of Muller cells (changes in b-wave amplitude) indicates a possible
difference in the mechanism of glucose metabolism of glia versus neurons. The electrophysiological effect of insulin under low glucose suggests its passage across the blood-retina barrier.
The increase in the standing potential is likely to be a receptor-mediated retinal pigment
epithelium effect. These results provide evidence in the retina for the reported multifunctional
nature of the insulin receptor. Invest Ophthalmol Vis Sci. 1997;38:792-799.
Current information about the action of insulin in
the retina is incomplete. Previous data indicated that
the uptake of glucose is insensitive to insulin in the
retina1 and in the brain.2 However, recent work has
shown that this peptide elicits various physiological,
developmental, and behavioral responses when administered into the brain or into specific central nervous system culture systems.3 Among the physiological
effects, an influence on visual-evoked potentials4 and
auditory brainstem responses5 has been reported in
humans. Research on the cellular actions of insulin
has shown varying effects on neuronal activity and dif-
Fxom the Neurophysiology-Lahoratmy, Department of Ophthalmology, University
Hospital, CH-8091 Zurich, Switzerland.
Submitted for publication July 11, 1996; revised October 21, 1996; accepted
December 13, 1996.
Proprietary interest category: N.
Refjrint requests: Giinter Niemeyer, Neurophysiology-Laboratory, Department of
Ophthalmology, University Hospital, CH-8091 Zurich, Stuilzerland.
792
ferential actions on the metabolism of glucose in glial
and in neuronal cells.6"8 Insulin receptors have been
identified in retinal cell populations of several mammalian species,9"12 and insulin-degrading glutathioneinsulin transhydrogenase has been localized in the
mammalian retina.13
We herein report on experiments addressing the
short-term effects of arterially administered fast-acting
insulin at postprandial and at higher levels on the
b-wave of the electroretinogram (ERG), optic nerve
response (ONR), standing potential (SP), and flow
rate in the isolated, arterially perfused cat eye14 under
normal conditions and during low glucose levels (corresponding to hypoglycemic conditions).
MATERIALS AND METHODS
Animals
The nine eyes used in the current study were enucleated from nine anesthetized adult female cats (pur-
Investigative Ophthalmology & Visual Sci<
Copyright © Association for Research in 1
e, April 1997, Vol. 38, No. 5
on and Ophthalmology
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Insulin Effects on Retinal Electrophysiology
chased from Ciba-Geigy, Basel, Switzerland, and from
IFFA CREDO, Lyon, France) in accordance with the
regulations of the cantonal veterinary authority of Zurich and with the ARVO statement for the Use of
Animals in Ophthalmic and Vision Research. Details
on the anesthetic methods15 and the experimental
technique of arterial perfusion of the in vitro cat
eye1416 have been published previously and are summarized here only.
Anesthesia
After premedication with atropine sulfate and ketamine hydrochloride (Ketasol; Graeub, Bern, Switzerland), the cats were anesthetized deeply with pentobarbital hydrochloride (Nembutal; Abbott, North Chicago, IL), paralyzed by gallamine triethiodide,
intubated, and respirated artificially (respirator 66IA;
Harvard Apparatus, South Natick, MA) with oxygenenriched (30%) room air. We used fentanyl (Fentanyl;
Janssen, Beerse, Belgium) and liquaemin (Liquemin;
Roche Pharma, Basel, Switzerland) before the enucleation of the first eye and induced death after the enucleation of the second eye (which was used for related
studies) with a bolus overdose of pentobarbital intravenously.
793
trials shown in Figure 1, by broad band chromatic
glass filters (Schott OG 590 or Schott BG 12; Schott,
Mainz, Germany). The ERG was recorded with salt
bridge Ag-AgCl electrodes positioned in die vitreous
and on the posterior scleral surface. The ONR was
recorded with a second pair of Ag-AgCl electrodes,
located at the cut end (suction electrode) and on die
distal surface of the nerve. The duration of light stimuli was 20 or 400 msec, delivered with an interval of
30 seconds. The perfusate flow rate, which reflects
total vascular resistance, was measured with an infrared drop-counter. Amplified (PARC 113; EG & G,
Princeton, NJ) and filtered (3750 filter; Krohn Hite,
Avon, MA) ERG and ONR signals were fed to a digital
oscilloscope (oscilloscope 4050; Gould, Cleveland,
OH), to a four-channel chart recorder (Gould RS
3400), to an FM magnetic tape recorder (Racal 7DS;
Racal Recorders, Southampton, England) and to a
personal computer. For data storage and off-line analysis, we used the LabVIEW for Windows 3.0 software
(National Instruments, Austin, TX), programmed by
Dr. A. Kaelin-Lang.18
Data Processing and Statistical Analysis
After cannulation of the ophthalmociliary artery, the
eye was perfused by constant hydrostatic pressure with
oxygenated glucose- and insulin-free tissue culture
medium (Medium 199; Bio Concept, Allschwil, Switzerland), which was buffered to reach pH 7.4 at 37°C.
The perfusate was supplemented with albumin 25
g/1 (Fluka, Buchs, Switzerland) and amikacin sulfate
63.9 fjM (Amikin; Bristol-Meyers Squibb, Princeton,
NJ). A pump (Harvard Apparatus 22; Harvard Apparatus South Natick, MA) was used for arterial application
of glucose at normal (5.5 mM = 99 mg/dl) or at
reduced concentrations of 2 and 1 mM for 8 minutes
and of fast-acting human insulin (Velosulin HM, a gift
from Novo Nordisk, Kiisnacht, Switzerland) for up to
60 minutes. Insulin was applied at a postprandial concentration of "»140 /LtU/ml,17 corresponding to «s5
ng/ml, and at levels of 10 and 20 times higher concentrations for up to 60 minutes.
Original traces were plotted with an XY-recorder
(Linseis LY 16100 II; Linseis, Selb, Germany) from
data stored on a Microsoft (Redmont, GA) personal
computer. Excel for Windows 4.0 and Sigma Plot for
Windows 2.1 (Jandel, San Rafael, CA) were used for
graphic illustrations and statistical analysis. Mean amplitude values of control signals recorded 5 minutes
before glucose and insulin challenges at steady-state
conditions (10 signals for the ERG and the ONR, 60
for the perfusate flow) were set to 100% (i.e., all data
were normalized). Changes in the SP are expressed
as changes from the control value in millivolts. An
effect in a single trial was considered as significant
when deviating from the mean normalized control
value by more than two standard deviations. Changes
in mean effects in different trials were evaluated with
the paired t-test (P < 0.05 accepted as significant),
including only the last seven signals at minutes 5 to 8
of a low-glucose challenge for the ERG and the ONR.
These data represented approximately steady-state
conditions with partial glucose deprivation (Fig. 3).
Other data are expressed as mean ± standard error
of the mean.
Stimulation, Recording, and Data Acquisition
RESULTS
Photic stimulation was performed in full dark adaptation by an optical system using a 150 W xenon lamp
(maximum intensity of 11.54 log quanta [507 nm]
deg~2 sec' 1 at the cornea) through a modified fundus
camera in a Maxwellian view. Light flashes were attenuated by neutral density filters and, except for the
The application of insulin in the arterially perfused cat
eye failed to affect light-evoked electrophysiological
responses recorded at standard glucose concentration. However, the reduction in b-wave amplitude during step decreases in glucose concentration down to
2 and to 1 mM was significantly greater in the presence
Arterial Perfusion, Low Glucose, and Insulin
Trials
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794
Investigative Ophthalmology & Visual Science, April 1997, Vol. 38, No. 5
B
700
70
control
effect (insulin 50ng/ml)
recovery
600 500
ERG b-wave
60 50 -
400 -
40 -
300 -
30 -
200 -
20 -
100
10 -
2
3
log relative I
control
effect (insulin 50ng/ml)
recovery
ONR-ON
3
4
5
log relative I
FIGURE i. Response amplitude versus stimulus intensity (V/logl) functions of the b-wave (A)
and the ONR-ON component (B) under dark-adapted stimulus conditions, indicated in
log relative intensities before [filled squares), during (open triangles), and after (filled circles)
application of insulin at 50 ng/ml, a concentration 10X higher than the postprandial level.
Stimuli were white light pulses of 400 msec duration. ONR-ON = optic nerve response,
ON-component.
of insulin. The low glucose-induced decreases in the
amplitude of the ONR-ON component, in contrast,
were not enhanced by insulin. Insulin led to a small
but reproducible and concentration-dependent increase in SP. No change in perfusate bulk flow in
response to insulin at normal or at reduced glucose
was observed.
Electroretinogram b-Wave and Optic Nerve
Response
Normal b-waves and ONRs were recorded in the absence of insulin (replacing the insulin-containing calf
serum19 in the perfusate by albumin; n = 9 eyes).
Under normal glucose concentration, insulin at postprandial and higher levels (n = 6 trials) failed to alter
the magnitude and time course of both the b-wave and
the ONR-ON amplitude by more than ±2 standard
deviation from the mean control value. No shift in the
V/logl function of b-wave and ONR-ON amplitude
could be observed during application of insulin at rodmatched conditions (n = 2 trials; Fig. 1).
In contrast, insulin significantly reduced the bwave amplitude during step decreases in glucose concentration down to 2 (P < 0.05) and 1 mM (P <
0.01), but did not alter the ONR-ON amplitude (n
= 8 trials; Fig. 2). The time course in changes of nor-
malized b-wave and ONR-ON amplitudes with corresponding original traces (insets) of single trials is depicted in Figure 3. The differential effect on the bwave compared to the ONR-ON amplitude was independent of the duration of the light stimulus of 20 or
400 msec (n = 2 trials). Increasing the insulin concentration in the same preparation from 5 to 100 ng/
ml did not enhance further the low glucose-induced
decrease in the ERG b-wave and left the ONR-ON
unaffected (Fig. 4). Recovery of the b-wave amplitude
after low glucose challenges to preinsulin values was
not observed in all trials within 30 minutes.
Three successive low glucose trials of 1 mM in
the same preparation yielded similar results (b-wave:
34.1%, 33.7%, 30.4%, maximum standard error of the
mean ± 2.1%; ONR-ON: 44.4%, 43.8%, 41.1%, maximum standard error of the mean ± 4.9%) when the
significant progressive enhancement of responses during the duration of the perfusion with low glucose was
excluded.
Standing Potential and Flow Rate of Perfusion
Insulin induced a small, but reproducible, increase in
SP (n = 8 trials). The changes induced by postprandial
insulin concentration ranged up to 0.25 mV. A concentration-dependent effect was found when insulin
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Insulin Effects on Retinal Electrophysiology
795
26 27
120
ONR-ON
ERG b-wave
o
insulin receptor. ' Yet, blood-born insulin appears
not to be required for maintaining normal retinal
electrophysiological responses within the periods (up
to 10 hours) investigated here.
b-Wave and Optic Nerve Response
Metabolic Versus Neuromodulatory Mechanisms of Insulin Action. Modulation of human visually evoked potential4 and auditory brainstem responses5 by insulin
have been shown within 20 minutes, and therefore the
duration of insulin application for up to 60 minutes in
our study should have been adequate to detect effects
on retinal function. However, a substantial modula•
low glucose (control)
tory action of insulin on the neurons that contribute
O low glucose + insulin
to the b-wave generation seems unlikely, because the
more proximally generated, exclusively neuronal
ONR virtually was unaffected by insulin.
2.0mM
1.0mM
2.0mM
1.0mM
Regarding the metabolism of glucose, evidence
glucose
has been obtained for the concept of a metabolic couFIGURE 2. Normalized average changes (n = 8 trials) of the
pling between neurons and glia whereby metabolites,
b-wave (left) and ONR-ON amplitudes (right) under 2 and primarily lactate produced from glucose by glial cells,
1 mM glucose, respectively. A value of 100% (filled circles)
provide the "fuel" for photoreceptors and probably
represents the mean value of amplitudes under low glucose
for other retinal neurons.28 In the rat, it has been
alone, and open circles indicate the corresponding mean
clamp, insulin supamplitudes in response to low glucose plus insulin (*P < reported that using euglycemic
29
0.05; **P < 0.01). Within the same preparation, effects of presses glucose utilization and decreases glucose oxi30
insulin on the electroretinogram b-wave amplitude under
dation in specific brain regions. However, insulin
low glucose, regardless the concentration of insulin, were
does not stimulate glucose metabolism in fetal chick
found to be homogeneous and results were pooled. Error
neurons,31 whereas insulin has been shown to increase
bars = ± SEM.
glycogen stores of astroglia-rich primary cultures from
rat brain32 and also in cultured rabbit Mtiller cells.33
In addition, insulin at a concentration of 10 ng/ml
was increased to levels 10 and 20 times higher than
stimulated D-glucose conversion to glycogen in endopostprandial concentrations (increases of as much as
thelial cells and in pericytes grown from retinal mi0.75 mV). The response showed a latency of several
crovessels.34 We conclude that the insulin-enhanced
minutes and exceeded the duration of the insulin apdecrease of the ERG b-wave obsen'ed here only under
plication.
low glucose likely is to be caused by the suppressant
Perfusate flow was affected neither by insulin apaction of insulin on the glucose use by Muller cells.
plication nor by its removal (n = 4 trials). Decreases
Under physiological glucose levels, retinal glia cells
in glucose concentration down to 2 or 1 mM for 8
tolerate the insulin-induced effect, and no electrominutes, alone or in combination with insulin, also
physiological changes become apparent. When reducfailed to induce significant changes in the vascular
ing glucose to levels that affect the signal amplitude
resistance measured at the level of the ophthalmociliper se, any suppression of glucose use would be exary artery (n = 6 trials).
pected to decrease the signal amplitude further.
DISCUSSION
Passage of Insulin and Glucose Across the
Blood-Retina Barrier
Using the in vitro arterially perfused cat eye, effects
of insulin on three parameters of retinal function of
different origin were monitored: the ERG b-wave, a
neuron-glia interaction response20"""1; the ONR, neuronal in origin14'23'24; and the SP, reflecting mainly the
voltage across the retinal pigment epithelium (RPE).25
The diversity of reactions of different retinal structures
to insulin, evident in the differential responsiveness
of the electrophysiological parameters, is in agreement with the reported multifunctional nature of the
In its function, the blood-retina barrier is comparable
to the blood-brain barrier.35'36 Correspondingly, the
glucose transporter GLUT1 has been localized in cells
of the blood-brain barrier37 and at the inner38 and
outer blood-retina barrier.38'39 Similarly, insulin receptors have been identified in endothelial cells of
brain microvessels,40'41 in retinal blood vessels,42 and
also on the basolateral membrane of the RPE.11 Evidence exists for both receptor-mediated and saturable
transendothelial transport of insulin across the blood-
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Investigative Ophthalmology & Visual Science, April 1997, Vol. 38, No. 5
m control (no insulin)
A 5ng/ml insulin
\ * r
_ T
-5
0
5
glucose 1 .OmM
80 -
ON amplitude
A
Z
O
15
20
25
time (min.)
• control (no insulin)
A 5ng/ml insulin
A
A
AB
••
A
*A
A
"
A
j A AA
A
•
A
•A
•
A
i
m
40-
control
^—"'^ lowglu
+ insulin
~••
A i
•
1 •
A
100 - A • B I B |
.A
A
A
d
\
"
A
A"
• •
control
i
•
V-" J
20 •
1
0 -5
+ insulin
i
i
0
5
glucose 1.0mM
l
l
10
15
20
25
time (min.)
FIGURE 3. Time course of changes in normalized b-wave (A) and ONR-ON amplitudes (B)
in response to 1 mM low glucose challenges without (filled squares) and with [open triangles)
insulin. Insets: (A) electroretinogram (ERG) and (B) optic nerve response traces. Each trace
represents the average of three signals at 5.5 mM glucose (control), at low glucose (thin trace),
and low glucose plus insulin [heavy trace). Light stimulus (OG 590) 400 msec. Electrical
filters ERG: 0.03 to 100 Hz; ONR: 0.1 to 100 Hz and 50 Hz notch filter. Calibration: ERG
100 fiV; ONR 20 //V; 100 msec for both. ONR-ON = optic nerve response, ON-component.
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Insulin Effects on Retinal Electrophysiology
43 4fi
brain barrier.
However, in an earlier study, insulin
was shown to bind to retinal endothelial cells without
penetrating into the retina.47 According to Pardridge
et al,43 insulin is transported across brain microvessels
with a t/2 of ~70 minutes and the amount of specifically bound insulin on microvessels reaches a 50%
saturation point at approximately 10 ng/ml. The enhancement by insulin of the low glucose-induced decrease in the amplitude of the b-wave, a signal generated within the neuroretina,20"22 offers electrophysiological evidence for the passage of insulin across the
blood-retina barrier. A saturable mechanism, as has
been characterized for the blood-brain barrier,
would have to be postulated, because increasing the
insulin level above the postprandial concentration did
not enhance the effect on the b-wave. In contrast, the
SP, generated across the RPE, moderately was insulinsensitive at normal glucose levels and showed a concentration-dependent increase. Insulin receptors, as
likely mediators of the increase in SP, could be identified on cultured human RPE cells by Takagi et al,12
who suggested a role of insulin in the regulation of
RPE functions. A concentration-dependent increase
in SP would not require the passage of insulin across
the blood-retina barrier in light of immunohistochemical analysis on embryonic rat eyes showing insulin receptors exclusively on the basolateral membrane.11
Interestingly, Honda48 reported an increase in amplitude of the b-wave and the oscillatory potentials in
response to high insulin concentrations in the isolated
rabbit retina. In that study, however, the lower insulin
effect of low
glucose (1mM)
effect of low glucose (1mM)
+ insulin
797
concentration, which still exceeded the postprandial
serum level by approximately 1000 times, failed to
induce changes in the ERG.
Unaffected Ocular Circulation
No consistent changes in perfusate flow rate were detectable in response to insulin, either at normal or at
low glucose levels. Insulin has been shown to hyperpolarize the membrane voltage of retinal pericytes and
might be involved in the regulation of the retinal microcirculation.49 However, small changes in retinal
perfusate flow hardly can be recorded at the level of
the ophthalmociliary artery, because the uveal flow in
the cat is approximately 40-fold higher and does not
necessarily respond in parallel to the retinal flow.50
Potential Function of Retinal Insulin Receptors
The presence and distribution of insulin receptors in
the mammalian retina (monkey photoreceptor and
neuronal cell bodies, human Muller cells9; human retina10; rat RPE cells11; cultured human RPE cells12)
suggest a functional significance for this peptide beyond the moderate metabolic effects shown here. Neuromodulatory actions, as shown for other parts of the
central nervous system, were not detected in the current electrophysiological investigation. A number of
studies have indicated that insulin exerts trophic and
developmental function in the nervous system.3'6'7'51'52
These effects, however, would exceed the time span
that can be studied in the perfused cat eye model.
Similarly, effects of insulin on a specific distribution
of glycogen stores in glial as well as in neuronal cells
of the cat retina53 and on glucose transporter systems,7
probably important for the retinal homeostasis of glucose,54 could neither be detected nor excluded in the
current study. Interestingly, insulin has been shown
to reduce ischemic brain damage independent of its
hypoglycemic effect, indicating a direct influence on
the brain.55
Key Words
electroretinography, hypoglycemia, insulin, optic nerve, retinal metabolism
Acknowledgments
insulin 5ng/ml
insulin 100ng/ml
FIGURE 4. Mean normalized amplitudes (last seven signals of
single trials) of the b-wave (open bars) and the ONR-ON
{hatched bars) at 1 mM low glucose alone (left side) and in
response to 5 and 100 ng/ml insulin (right side), respectively.
A value of 100% = mean amplitude of the respective signal
at 5.5 mM glucose. Error bars = ±standard error of the
mean.
The authors thank Mrs. G. Gantenbein for competent technical assistance, Dr. U. Gerber (Brain Research Institute,
Zurich, Switzerland) for helpful comments during the preparation of the manuscript, and Drs. P. Autenried and H.
Thomas from the Laboratory Animal Unit of the University
of Zurich.
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