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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 Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933200/ on 05/05/2017 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 Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933200/ on 05/05/2017 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 Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933200/ on 05/05/2017 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- Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933200/ on 05/05/2017 796 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. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933200/ on 05/05/2017 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. References 1. Keen H, Chlouverakis C. Metabolic factors in diabetic retinopathy. In: Graymore CN, ed. Biochemistry of the Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933200/ on 05/05/2017 798 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 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