Electrophysiological and Metabolic Characterization of - Diabetes

Electrophysiological and MetabolicCharacterization of Single p-Cells andIslets From Diabetic GK RatsS.J. Hughes, M. Faehling, C.W. Thorneley, P. Proks, F.M. Ashcroft, and P.A. Smith

We have used the whole-cell recording technique todetermine whether ATP-sensitive potassium (K jp) cur-rents, voltage-dependent Ca2+ currents, and exocytosisare different in single JS-cells from pancreatic islets ofGoto-Kakizaki (GK) rats, a novel model of NIDDM, andnormal rats. In addition, we have also measured theinsulin secretory responses, ATP content, and the rateof glucose metabolism in intact islets. Although theglucose sensitivity of the KATP current was similarbetween GK rats and controls, in the absence of glucose,KATP current density was larger in GK rats, whichresulted in a more hyperpolarized membrane potential.Whole-cell Ca2+ currents were similar. By monitoring thecell capacitance with a fixed intracellular solution, nodifference was detected in the exocytotic responses ofP-cells from normal and GK rats. In islets from GK rats,the rates of glucose utilization ([3H]H2O productionfrom 5-[3H]glucose) and oxidation ([14C]CO2 produc-tion from U-[14C]glucose) were not significantly differ-ent from controls. Insulin secretion, however, wasimpaired (by 50%), and this was paralleled by a smallerincrease in ATP content in response to stimulation by10 mmol/1 glucose in islets from GK rats when com-pared with controls. Under conditions in which KATPchannels were held open and the effects of glucosewere independent of membrane potential, insulinrelease was still significantly lower in GK rat isletsthan in controls. These findings suggest that theimpaired insulin secretion in islets from GK rats doesnot simply result from a failure to close KATP channels,nor does it result from an impairment in calcium secre-tion coupling. Diabetes 47:73-81, 1998

In patients with NIDDM, both glucose-induced insulinrelease and the actions of insulin at target tissues areimpaired (1,2). The mechanisms underlying thesedefects remain unclear. The Goto-Kakizaki (GK) rat

represents a novel spontaneous model of NIDDM without

From the Department of Physiology (S.J.H., C.W.T.), Division of Basic Med-ical Science, Imperial College School of Medicine at St. Mary's, London; theDepartment of Physiology (M.F., F.M.A., P.A.S.), University of Oxford,Oxford, England, U.K.; and the Institute of Molecular Physiology andGenetics (P.P.), Bratislava, Slovakia.

Address correspondence and reprint requests to P.A. Smith, the Depart-ment of Physiology, University of Oxford, South Parks Rd., Oxford, 0X1 3PTU.K. E-mail: [email protected].

Received for publication 16 December 1996 and accepted in revisedform 10 September 1997.

GK, Goto-Kakizaki; KATP, ATP-sensitive potassium; TEAC1, tetraethy-lammonium chloride.

obesity (3) and was derived by using impaired glucose toler-ance as a selection index over 35 generations. The basis of thediabetes is polygenic (4,5), and multiple abnormalitiesinvolved in the mechanisms of glucose homeostasis arelikely. Using this model, several studies have been conductedto identify defects in the mechanism of insulin release (6-11).It was recently proposed that the impairment of glucose-induced insulin secretion in GK rats may be due, at least inpart, to a deficiency of flavin adenine dinucleotide-linkedglycerophosphate dehydrogenase in islets of Langerhans(12). It was suggested that the reduced activity of the glycerolphosphate shuttle was responsible for the reduction in theratio of the rate of oxidation/utilization of glucose alsoobserved in islets of these animals (13). It has also beenreported that ATP-sensitive potassium (KATP) channelsexhibit a lower sensitivity to glucose in GK rat p-cells, whichmay reflect a reduction in glucose metabolism (9). Our pre-liminary finding that glucose metabolism is not impaired inislets from GK rats (14) prompted us to determine whetherthe glucose sensitivity of K^p channels is altered in our GKrats and if not, what other pathways may account for theirdefective insulin secretory responses. To this end, we havealso investigated whether differences exist between Ca2+

currents and the secretory responses at the single cell levelin p-cells from GK rats compared with controls.

RESEARCH DESIGN AND METHODSMaterials. Bovine serum albumin (BSA), hexokinase, glucose-6-phosphate dehy-drogenase, ATP, and NADP were supplied by Boehringer-Mannheim (Lewes,U.K.). Collagenase Type 1A, Hanks' balanced salt solution (HBSS), amphotericinB, diazoxide, U-[14C]glucose (370 mCi/mmol), and methyl-benzethonium hydrox-ide were all supplied by Sigma Chemical (Poole, U.K.). [125I]Insulin (100 uCi/ug)was supplied by IifeScreen (Watford, U.K.), and 5-[3H]glucose (15.7 Ci/mmol) wassupplied by Amersham International (Aylesbury, U.K.). Coomassie Blue dyereagent for protein determination was supplied by Biorad Labs (Watford, U.K.).The ATP monitoring kit was supplied by Bio-Orbit (Turku, Finland). Sagatal wassupplied by Southern Veterinary Supplies (Lewes, U.K.). Anti-rat insulin anti-body was a gift from Dr. R. Turner, University of Oxford, Oxford, U.K. Tissue cul-ture reagents were supplied by Gibco (Paisley, U.K.). All other reagents used weresupplied by Sigma Chemical.

Animals. A colony of GK rats was established at the animal facility of St. Mary'sHospital Medical School in September 1992 from two male and seven female ani-mals supplied by Dr. K.-I. Suzuki from Tohoku Medical School, Sendai, Japan.Female rats were selected at 14 weeks of age or older for the experiments outlinedbelow. Controls were age-matched Wistar rats supplied by Harlan Olac. All animalswere allowed free access to food and drink. The nonfasting blood glucose con-centrations of the animals used in this study were 11.3 ± 0.3 (n = 13) for controlsand 19.4 ± 0.9 (n = 17) for GK rats determined by the Hexokinase method (15).Islet isolation and single cell preparation. Rats were anaesthetized withSagatal (1.5 ml/kg body wt, intraperitoneal [i.p.]) and killed by exsanguination.Islets of Langerhans were prepared by collagenase (16). For electrophysiologicalexperiments, islets were dispersed into single cells as previously described (17),

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plated onto plastic petri dishes, and cultured for 18-36 h before study. Islets anddispersed cells were cultured in RPMI1640 tissue culture medium containing 11mmol/1 glucose, glutamine, 10% (vol/vol) fetal calf serum, and antibiotics at37°Cin a humidified atmosphere of 5% CO2,95% air. The protein content of samples ofislets collected was determined using the Biorad method (18).Glucose metabolism in isolated islets. The rate of glucose metabolism wasmeasured in groups of 10 islets in HEPES-buffered Krebs-Ringer buffer con-taining 2 mg/ml BSA and various glucose concentrations. Glucose utilization andglucose oxidation were measured using 5-[3H]glucose and U-[14C]glucose, respec-tively, as previously described (19).

Electrophysiological measurements in dispersed islet cells. Whole-cellcurrents and membrane potentials were recorded using the perforated-patchconfiguration of the patch-clamp technique, which retains metabolism intact(18,20). Most experiments were carried out at room temperature (22-25°C). Volt-age errors due to liquid junction potentials (<5 mV) and the series resistance (<3mV) were small and have been ignored.

For measurement of KATP currents, cells were continuously superfused witha modified Hanks' solution. The pipette was tip-filled with a solution that contained70 mmol/1 KjSO,,, 10 mmol/1 NaCl, 10 mmol/1 KC1, 1 mmol/1 MgCL,, 10 mmol/1HEPES (pH 7.4), 1 mmol/1 EGTA, and 40 mmol/1 sucrose and then back-filled withthe same solution supplemented with 0.1 mg/ml amphotericin B (20). Whole-cellKATP currents were measured as the current generated by a pulse protocol of alter-nate ± 10 mV steps of 200-ms duration applied at a frequency of 0.5 Hz from a hold-ing potential of -70 mV. The currents elicited by this protocol are carried solelyby K^p channels (17,20). For construction of the glucose dose-response rela-tionship, KATP currents measured during steady state were normalized to currentsrecorded in the prestimulatory control (glucose-free) solution.

For the measurement of Ca2+ currents, the pipettes contained a similar solutionto that used to study K^p currents, but with I^SO,, replaced by Cs2SO4 to block out-ward K+ currents. For these studies cells were superfused with a solution con-taining, 5.6 mmol/1 KC1,1.2 mmol/1 MgCl2,10 mmol/1 HEPES (pH 7.4 with NaOH),5.5 mmol/1 glucose, 30 mmol/1 tetraethylammonium chloride (TEACl) with either118 mmol/1 NaCl and 2.6 mmol/1 CaCl2 or 108 mmol/1 NaCl and 10 mmol/1 BaCL,.As a substitute for Ca2+, 10 mmol/1 Ba2+ was used because it screens a similaramount of membrane surface charge as 2.6 mmol/1 Ca2+ and consequently will main-tain the same voltage-dependence of Ca2+-channel gating (21). Glucose was pres-ent to maintain the calcium currents (22) and to also block KATP currents (17,20).TEACl was added to block any residual ion movement through K+-channels. Cur-rents flowing through Ca2+ channels were, in most studies, evoked from a holdingpotential of-70 mV by incremental 10-mV voltage steps of 200-ms duration. Thesewere applied at a frequency of 0.2 Hz to prevent intracellular Ca2+ loading. To cor-rect for linear leak currents and uncompensated capacity transients, the currentresponses to consecutive negative voltage steps of -100, -90, and -80 mV werescaled, averaged, and subsequently subtracted from the raw current traces.

Peak values of Ca2+ currents were determined as the maximum of a runningaverage (10-ms window), steady state values were taken as the average of the last50 ms of the inward current. The inactivating component of the inward currentwas approximated as a function of time, t, by a single exponential of the form, aexp(-£/\), where a is the peak amplitude at t = 0 (pA) and \ is the time constantof decay (ms).Secretion from single cells. Secretion was measured at the single cell level byrecording the increase in cell electrical capacitance that occurs as secretoryvesicles insert into the plasma membrane. For these studies single 3-cells werevoltage-clamped at -70 mV using conventional whole-cell techniques. Secretionwas induced by the Ca2+ influx associated with the activation of the voltage-gated Ca2+ channels by voltage pulses to +20 mV of 500-ms duration. Capacitancechanges were measured as previously described (24). For these studies, the bathwas continuously perfused at 31-32°C with the calcium-containing solutionabove. The pipette solution contained (in millimoles per liter): 125 K-glutamate,10 mmol/1 KC1, 10 mmol/1 NaCl, 1 mmol/1 MgCL,, 10 mmol/1 HEPES, 50 umol/1BAPTA, 3 mmol/1 ICjATP, 0.1 mmol/1 Li2GTP (pH 7.15 with CsOH).Insulin release and ATP determination. Insulin release in static incubationswas measured in groups of five islets in tubes containing 0.6 ml of HEPES-buffered Krebs Ringer, 2 mg/ml BSA, various glucose concentrations, and otheradditions as indicated. Incubations were stopped after 1 h at 37°C by cooling onice and after centrifugation aliquots were collected for insulin assay by radioam-munoassay (RIA) (25). In parallel experiments, groups of five islets were incubatedin 0.1-ml volumes of medium for 1 h at 37°C. Incubations were stopped by addi-tion of 50 ul 10% (vol/vol) perchloric acid, followed by rapid freezing. After thaw-ing, the samples were sonicated and neutralized with 10 mol/1 NaOH. The ATP con-tent of the islets was then determined using a Bio-Orbit luciferin-luciferase kit onan LKB luminometer (26).Statistical analysis. Data are means ± SE for the number of experiments indi-cated. Statistical analysis, where appropriate, was carried out using the Student'st test.

RESULTS

Glucose metabolism. The effect of changing the mediumglucose concentration on the rate of glucose utilization andoxidation in islets is shown in Table 1. Raising the medium glu-cose concentration to 10 mmol/1 significantly increased therates of glucose utilization and oxidation in islets isolated fromcontrol and GK rats, respectively. In islets from GK rats, therates of glucose utilization and oxidation were not significantlydifferent from controls at all glucose concentrations tested.Electrophysiological measurementsKpxp currents. Figure \A shows a representative recordingof whole-cell K^p currents from a normal rat 3-cell. In theabsence of glucose, the current amplitude was stable. Appli-cation of the sulphonylurea tolbutamide (0.2 mmol/1) pro-duced a rapid and reversible block of the whole-cell current,confirming that most of the resting current flows throughK^p channels. In 3-cells from normal and GK rats, 0.2 mmol/1tolbutamide inhibited the current by 94 ± 2% (n = 3) and 89± 8% (n = 3), respectively. Addition of 2 mmol/1 glucoseblocked 27 ± 20% (n = 5) and 58 ± 30% (n = 6) of the K^p cur-rent in (3-cells from normal and GK rats, respectively (Figs.IB,C), this difference was not significant (P = 0.3). Althoughin Fig. 1, the displayed time course of block of the K^p cur-rent by 2 mmol/1 glucose is slower in the GK than in the con-trol rat, this difference was not significant (P = 0.28). The half-time of block was 107 ± 31 s (n = 4) and 130 ± 22 s (n = 6) innormal and GK rats, respectively. The time course of block ofthe K pp current produced by 5 mmol/1 glucose (Fig. 2) was,however, significantly slower (P = 0.03) in GK rats than thatmeasured in controls: The half-time of block was 129 ± 20 s(n = 4) and 72 ± 11 s (n = 4) in GK and control rats, respec-tively. In both types of rat, 5 mmol/1 glucose almost com-pletely blocked the K ^ current (Figs. 2A, E), the K ^ currentbeing blocked by 78 ± 3 (n = 7) and by 80 ± 5 % (n = 8) in con-trol and GK rat, respectively.

The relationship between glucose concentration and theextent of current inhibition was investigated by alternatingeach glucose test solution with a glucose-free control solution.Figure 3A shows the mean glucose dose-response curves forthe K pp current elicited by a -i-10-mV voltage pulse in (3-cellsfrom normal and GK rats. In GK rats, the amplitude of thewhole-cell K^p current elicited in the absence of glucosewas significantly larger (P = 0.02) at 49 ± 10 pA (n = 11), thanthat measured in controls, 24 ± 4 pA (n = 16). To exclude thepossibility that this difference arose because of variations inP-cell size, we further compared the density of the K^p cur-rent, defined as a ratio of the current amplitude to the cellcapacitance. Although the cell capacitance varied consider-ably from cell to cell, the mean cell capacitance measured inperforated-patch experiments in GK rats (3.9 ± 0.2 pF, n = 20)was not significantly different (P = 0.23) from that in fJ-cellsfrom controls (4.2 ± 0.2 pF, n = 39). Consequently, the K ^ cur-rent density in p-cells from GK rats at 0 mmol/1 glucose (11± 3 pA/pF, n = 10) was still significantly (P = 0.03) greater thanthat in controls (4.6 ± 1 pA/pF, n = 13).

The K pp current was blocked with increasing glucose con-centrations in cells from both strains of rat. Although at lowglucose concentrations the magnitude of K^p current washigher in the GK rats compared with normal subjects, this dif-ference was, as we have just shown, only significant at 0mmol/1 glucose. On first glance, these data suggest that the glu-cose sensitivity of the inhibition of the K ^ currents in GK rats

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S.J. HUGHES AND ASSOCIATES

TABLE 1Glucose metabolism in islets isolated from control and GK rats

2 mmol/1 glucose6 mmol/1 glucose10 mmol/1 glucose

Oxidation (pmol •Control

2.8 ± 0.210.3 ± 0.713.1 ± 0.8

islet"1 • h"1)GK

3.1 ± 0.49.4 ± 0.9

11.4 ±1.1

Utilization (pmol •Control

12.2 ± 1.230.5 ± 3.641.1 ±6.2

islet"1 • h"1)GK

12.1 ±2.027.5 ± 5.628.4 ± 6.5 NS

Groups of 10 islets isolated from control or GK rats were incubated for 2 h at 37°C with various concentrations of glucose and 5-[3H]glucose and U-[14C]glucose to measure glucose utilization or oxidation, respectively. Data are picomoles produced per islet perhour and represent the mean ± SE of 10-12 observations from three to four separate experiments.

may be impaired; however, calculating the percentage inhibi-tion by expressing the K^p current amplitude in the presenceof glucose as a percentage of its mean current amplitude in thepreceding control solution demonstrates this is not the case(Fig. 3£): The glucose sensitivities of the inhibition of theKATP current are similar in the two strains of rats. Figure 3B alsoshows a threshold phenomenon; glucose concentrations <l-2mmol/1 fail to block the K ^ current, whereas a small increasein the glucose concentration to >2-3 mmol/1 produces amarked inhibition of the current. Any empirical model that isused to quantify such data must be able to reproduce this phe-nomenon. To account for the threshold phenomenon, a mod-ified Hill equation was fitted to the data as follows: when theglucose concentration (mmol/1), [G], is greater than thethreshold glucose concentration (mmol/1), [Go], at which theKATP channels are blocked, the % inhibition = 100 - 100 /{I +[([G] - G6)/(ECm - G6)]n). Otherwise, if [G] is less than [Go],the % inhibition = 0. EC^ is the glucose concentration that pro-duces half-maximal inhibition of K^p, and n is the Hill coef-ficient. The mean ECm values were not significantly differentbetween the two strains: 2.4 ± 0.2 mmol/1 (n = 6) and 2.6 ± 0.3mmol/1 (n = 7) for control and GK rats, respectively. Values ofGo and n were, 2.2 ± 0.3 mmol/1 (n = 6) and 0.36 ± 0.1 (n = 6)for normal, 2.2 ± 0.3 mmol/1 (n = 8) and 0.49 ± 0.1 (n = 7) forGK rats, respectively.

The EC50 values for the block of the K ^ current by glucosewe obtained using the perforated-patch whole-cell techniqueare consistent with those previously determined from bothcell-attached recordings of single K^p channel activity in rat3-cells (27) and from K+ efflux studies in intact islets (28).

In the absence of glucose, the resting potential of (B-cellsfrom the GK rat was significantly (P < 0.04) more hyperpo-larized (-71 ± 2 mV, n = 6) than that measured in control ani-mals (-66 ± 2 mV, n = 10). When a substantial proportion ofthe K^p channels were blocked by 5 mmol/1 glucose, theresting potential of GK rat (3-cell was still significantly (P <0.03) more hyperpolarized (-64 ± 3 mV, n = 3) than that meas-ured in normal subjects (-55 ± 2 mV, n = 5). At higher glucoseconcentrations, (3-cells from both strains of rat exhibitedcontinuous spiking electrical activity (data not shown).Calcium currents. Figure 4 shows that inward Ca2+ cur-rents were elicited in 3-cells from both normal and GK ratsat potentials positive to -50 mV. The magnitude and timecourse of the Ca2+ currents were very similar in both types ofrats; currents peaked at +20 mV and reversed at potentialspositive to +50 mV (Fig. 4). The currents displayed inactiva-tion, which was maximal at voltages around the peak voltage(Figs. 4A, B, and E). The rates of inactivation were quite vari-

able. The inactivating component of the inward Ca2+ current,at +20 mV, had an amplitude (a) of-29 ± 7 pA and time con-stant (\) of 30 ± 2 ms (n = 7) in normal rats, and an a of -24± 7 pA and X. of 36 ± 3 ms (n = 10) in GK rats. Neither of thesevalues were significantly different between the two strains ofrat. The presence of inactivation is suggestive of Ca2+-depen-dent inactivation, which is associated with L-type Ca2+ chan-

A 0.2 mmol/1 Tb

20 pA

B

V 'i .'i|* *" ' •» . '»» ' . '.-".'irh-

1 min

2 mmol/1 glucose

20 pA

1 min

2 mmol/1 glucose

40 pA

1 min

FIG. 1. Representative recordings of perforated-patch whole-cell KATP

currents from single rat p-cells. Currents were elicited by alternate±10-mV voltage steps from a holding potential of -70 mV. The inter-ruptions marked by arrows are when the amplifier was switched to cur-rent clamp to measure the membrane potential. A: effect of 0.2 mmol/1tolbutamide on a normal rat (J-cell. Note the expanded timescale andthe rapid block by the sulphonylurea. Effect of 2 mmol/1 glucose on ap-cell from a normal (.8) and a GK (C) rat.


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STUDIES OF SINGLE li-CELLS FROM GK RATS

A 5 mmol/l glucose A

,0pA

B1 min

5 mmol/l glucose

40 pA

1 min

FIG. 2. Representative effects of 5 mmol/l glucose on whole-cell KATPcurrents recorded with the perforated-patch technique in single P-cellsfrom a normal 04) and GK (.B) rat. Currents were elicited by alternate±10-mV voltage steps from a holding potential of-70 mV. The inter-ruption marked by the arrow is when the amplifier was switched to cur-rent clamp to measure the membrane potential.

nels and/or voltage-dependent inactivation of T-type Ca2+-channels, the latter which are also found in p-cells fromSprague-Dawley rats (21,23,29). In P-cells from both strainsof rat, replacement of Ca2+ with Ba2+ as the charge carrierincreased the magnitude of the inward currents almosttwofold and removed a substantial proportion of the inacti-vation, leaving voltage-dependent inactivation, which weattribute to the presence of T-type calcium channels (Figs. 4C,D). The inward Ba2+ currents recorded in p-cells from GKrats possess very similar amplitudes and time courses withalmost identical peak and steady state current-voltage rela-tionships to those measured in p-cells from control animals(Figs. 4C, D,F).

To obtain estimates of the relative contribution of T-typeand L-type Ca2+ channels to the macroscopic current, thefollowing experiments were performed. To abolish currentflowing through L-type Ca2+-channels, 100 umol/1 nifedipine,a specific dihydropyridine antagonist of these channels,which is not expected to affect T-type channels (30), wasadded to the bath. In both rat strains, the drug eliminated thesteady state current component leaving only the transient,inactivating current component, which we attribute to T-typeCa2+ channels (Fig. bA).

To remove current flowing through T-type Ca2+ channels,the holding potential was depolarized from -90 to -40 mV.Although this protocol has been reported to cause slightinactivation of L-type Ca2+ channels in P-cells from humans,it has been shown to selectively inactivate T-type but not L-type Ca2+ channels in cell-attached patches on single p-cellsfrom Sprague-Dawley rats (29). This protocol abolished thetransient, inactivating component of the Ba2+ current in bothtypes of rat (Fig. 5B). We attribute the remaining non-inacti-

U

75 -i

5CH

25 i

•fil nft ,—.IBI

3 5 10Glucose (mmol/l)

20

B

20-

1 40 HISc 60H

80-

100-5 10 15

Glucose (mmol/l)20

FIG. 3. Mean glucose dose-response curves for the inhibition of KATPchannel activity in P-cells. Expressed as (A) the KATP current elicitedby +10 mV from normal (open columns) and GK rats (shadedcolumns), and as (£) inhibition as a percentage of control, in normal( • ) and GK (O) rats. The lines in (£) are fits of the equation of % inhi-bition = 100 - 100 /{1+[([G] - Goy(EC50 - Go)]"}, to the mean data fromnormal rats (solid line) with Go = 1.9 mmol/l, EC50 = 2.2 mmol/l, andn = 0.5 (n = 6) and to the mean data from GK rats (dotted line) withGo = 1.5 mmol/l, EC50 = 2.0 mmol/l, and n - 0.7 (n = 7). *Significant dif-ference (P < 0.05).

vating current component to L-type Ca2+ channels. Takentogether, these data support the presence of both T- and L-typeCa2+ channels in p-cells from normal and GK rats.

Figure 5C shows the peak current-voltage relationshipsfor the nifedipine-insensitive inward Ba2+ currents. The datasuggest that the current flowing through T-type Ca2+ channelsis larger in the GK than in control rats; however, this differ-ence is not significant. This notion is also supported by theobservation that the inactivating component of the Ba2+ cur-rent, elicited by a voltage step to +20 mV from a holdingpotential of-90 mV, is also larger in amplitude in GK rats (-41± 10 pA, n = 7) than that measured in control animals (-28 ±7 pA, n = 6), although, again, this difference is not significant.The time constant of the decay of the Ba2+ currents elicitedat +20 mV are similar between the two species of rat: 100 ±18 (n = 6) and 105 ± 31 ms (n = 7) in control and GK rats,respectively. Comparison of the peak current-voltage rela-tionships for the non-inactivating Ba2+ current (Fig. 5Z)),elicited from a holding potential of-40 mV, suggests that theamount of L-type Ca2+ current is similar in p-cells from bothGK and control rats.

A rise in intracellular calcium is a key event in insulinsecretion coupling; integrating the area under the calcium cur-


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A

40 pA

B A

100 ms

c D

p Vm (mV)

-60 -40 -20 0 20 40

-100 -L

FIG. 4. Representative perforated-patch whole-cell recordings ofinward currents from single rat p-cells. Currents were elicited by 250ms pulses from a holding potential of -70 mV to potentials of -30, -20,0 and •(• 10 mV (top traces) and +20, +30, +40, and +50 mV (lowertraces). A: Ca2+ currents recorded from normal rat. B: Ca2+ currentsrecorded from GK rat. C: Ba2+ currents recorded from normal rat andD: Ba2+ currents recorded from GK rat. Note difference in scale to cur-rents recorded with Ca2+. Dotted lines show holding current level. E: cur-rent-voltage relationships for Ca2+ currents. F: current-voltage rela-tionships for Ba2+ currents. Mean peak ( • ) and steady state ( # ) valuesfrom normal rats (« = 9), mean peak ( • ) and steady state (O) valuesfrom GK rats (n = 13-16). Error bars have been omitted for clarity.

rents gives an estimate of the total calcium entry during thepulse and is an indication of the cell's fitness to elicit secre-tion. The total calcium entry during a voltage step to +20 mVwas 12 ± 1.5 pC (n = 9) in normal p-cells and was not signi-ficantly different at 14 ± 1.7 pC (n = 16) in GK rats.Exocytosis from single P-cells. Because there appeared tobe little difference in the properties of inward currents andassociated calcium entry in p-cells from normal and GK rats,we investigated the possibility that the secretory response tothe calcium influx may be impaired in p-cells from the GK rat.Studies using permeabilized islets have shown that calcium-dependent insulin secretion in islets from GK rats is normal(10); therefore we tested if this was also the case at the levelof the single P-cell. Although secretion studies from islets cangive information about total insulin secretion and also the rateof secretion, the latter is on the timescale of minutes, whereany temporal difference in early events may not be resolved.

B

40 pA

100 ms

Vm (mV)

-60 -40 -20 0 20 40 60

DVm (mV)

-60 -40 -20 0 20 40 60

U

-60 -1-

FIG. 5. Representative perforated-patch whole-cell recordings of Ba2+

currents in single P-cells from normal (left traces) and GK (righttraces) rats. A: currents elicited by a 250-ms pulse to +20 mV from aholding potential o f - 9 0 mV, before (larger trace) and after (smallertrace) bath addition of 100 umol/1 nifedipine. B: currents elicited bya 250-ms pulse to +20 mV from holding potentials of -90 mV (largertrace) and -40 mV (smaller trace). Dotted lines show holding currentlevel. C: mean peak current-voltage relationships for nifedipine-insensitive Ba2+ currents from normal ( • , n = 2) and GK ( • , n = 3) rats;error bars show standard error of the mean. D: mean peak current-volt-age relationships for Ba2+ currents elicited from a holding potential of-40 mV in normal ( • , n = 3 ) and GK rats (O, n = 10); because of thesimilarity, the error bars have been omitted for clarity.

Single-cell measurements of secretion, however, can yield use-ful temporal information about early events in secretion onthe tens of milliseconds timescale. Furthermore, studies at thesingle-cell level are free from paracrine influences that are pos-sible in the intact islet from neighboring endocrine cells.

For this series of experiments, we used the standardwhole-cell configuration, allowing us to control intracellularATP levels. To allow equilibration of the cell with the pipettesolution, secretion was studied after ~1 min of establishingthe whole-cell configuration. The average whole-cell capac-itance was 3.3 ± 0.2 pF (n = 20) in control and 3.3 ± 0.2 pF (n= 20) in GK rats. As a measure of secretion in individual P-cells, we chose the average of the exocytotic responsesevoked by three consecutive 500-ms voltage pulses from -70to +20 mV applied at a frequency of 0.03 Hz. This methodgreatly reduces pulse-to-pulse variation in secretion. In 95%of all cells tested, an increase in cell capacitance occurredafter membrane depolarization, followed by a slow decreaseattributed to endocytosis (Fig. 6). The average capacitanceincrease was not significantly different (P = 0.39) between nor-mal and GK rats, being 66 ± 10 fF/pF (n = 20) and 53 ± 13 fF/pF(n = 20), respectively. The mean whole-cell Ca2+ current


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STUDIES OF SINGLE K-CELLS FROM GK RATS

control GKrat

+20 mV-70 mV

+20 mV-70 mV

100 fF

0.5 nS 0.5 nS

10s 10 s

FIG. 6. Representative standard whole-cell recordings of membrane capacitance (middle traces), elicited by a 500-ms pulse to +20 mV from aholding potential of-70 mV, in normal (left trace) and GK (right trace) rats. The small positive pulse at the beginning of each capacitance recordis a calibration pulse of +100 fF. Top trace of both records shows the voltage protocol. Bottom traces are the corresponding membrane con-ductance record to illustrate the absence of cross-talk between changes in capacitance and membrane conductance.

amplitude and Ca2+ charge entry obtained during the pulsewere 26 ± 2.7 pA/pF (n = 14) and 4.5 ± 1.4 pC/pF (n = 13) innormal rat p-cells and 23 ± 1.3 pA/pF (n = 14) and 3.7 ± 0.7pC/pF (n = 13) in GK rat (3-cells, respectively. Neither theamplitude nor charge entry of the Ca2+ currents were signifi-cantly different between the two strains of rat (P > 0.3 and P> 0.6, respectively).Islet insulin release and ATP content. Insulin secretionfrom islets of GK rats incubated in medium containing 10mmol/1 glucose was significantly 50% lower than that of con-trol islets (Table 2). Release under basal conditions (2mmol/1 glucose) was similar in control and GK rat islets.Inclusion of 250 umol/1 diazoxide completely inhibited glu-cose-stimulated insulin release in all islets, In the presenceof 10 mmol/1 glucose, 40 mmol/1 K+, and 250 jimol/1 diazox-ide (i.e., under condition in which K^p channels were heldopen and the effects of glucose were independent of mem-brane potential), insulin release was still significantly lowerin GK rat islets than in controls. For comparison with elec-trophysiological experiments, insulin release was measuredin control and GK rat islets maintained in tissue culture for24 h. Glucose-stimulated insulin release (at 10 mmol/1 glu-cose) remained significantly higher (P < 0.001) in controls(320 ± 21 uU insulin • islet"1 • h"1, n = 8) than in GK rat islets(47 ± 6 uU insulin • islet"1 • h"1, n = 8). In freshly isolated islets,raising the medium glucose concentration from 2 to 10mmol/1 significantly increased the ATP content by 61% incontrols. Although the ATP content increased by 37% in GKrat islets incubated in 10 mmol/1 glucose, this increase wassignificantly smaller than that found in controls.

The protein and insulin content of freshly isolated isletswere 0.36 ± 0.02 (n = 8) and 0.34 ± 0.02 (n = 5) ug protein perislet and 2.4 ± 0.2 (n = 6) and 3.1 ± 0.3 (n = 5) mU insulin perislet in control and GK rats, respectively.

DISCUSSION

It has been shown in several laboratories, including this one,that insulin secretion in response to glucose stimulation is

impaired in adult GK rats both in vitro (6,7,9,31,32) and in vivo(5,29,33,34). Several studies have addressed the mechanismunderlying the impaired insulin secretory response. It hasbeen shown that the ability of GK rat islets to oxidize glucoseis either impaired (7,13) or normal (6), whereas glucose uti-lization is either normal or elevated (7,6). It was suggested thatthe reduction in the ratio of the rate of oxidation/utilization ofglucose resulted from an impairment in the FAD-linked glyc-erol phosphate shuttle (12), an enzyme complex responsiblefor transferring reducing power into the mitochondria Closureof K pp channels by glucose is a key event in insulin secretioncoupling in (3-cells (39). Tsuura et al. (9) compared the glucose-dependent block of K pp channels in cell-attached patchesfrom normal and GK rat p-cells. They concluded that inhibi-tion of channel activity occurred with a lower sensitivity to glu-cose in GK rat P-cells, whereas the activity and the propertiesof single K^p channels were normal (9).

In experiments presented here, we show that the rates ofboth glucose utilization and oxidation in islets from adult GKrats are similar to those in control islets. We also show that glu-cose closed K pp channels in GK rat (3-cells with a similar sen-sitivity to that observed in control cells. The reason for the dis-crepancy between our findings and the previously publishedstudy on K ^ channel function in pancreatic (3-cells of GK ratsis unclear. It may reflect the polygenic nature of the diabetesof GK rats since different colonies of these animals may havedifferent genotypes and hence different defects in insulinsecretion. A comparison of chromosomal mapping in twostudies (35) indicates similar as well as distinct genetic lociassociated with diabetes in two different colonies of GK rats(5,36). Furthermore, we used female rats that were >14 weeksof age, whereas Tsuura et al. (9) used male rats that were 8-12weeks old. Whether these age and sex differences contributeto our dissimilar results to those published is not known.

Our observation that the K^p current density is greater in(3-cells from GK rats than controls at nearly all glucose con-centrations tested could be misinterpreted as a decreased glu-cose sensitivity of the K^p current in the diabetic rats. Nor-


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TABLE 2Insulin secretion and ATP content in islets isolated from control and GK rats

Insulin release (uU • islet • h )Incubation medium Control GK

ATP content (pmol/islet)Control GK

2 mmol/1 glucose10 mmol/1 glucose10 mmol/1 glucose + diazoxide10 mmol/1 glucose + 40 mmol/1 K+/diazoxide

6.4 ± 1.5312 ± 20*10.2 ± 2.7205 ± 16.2

10.4 ± 2.6 NS151 ± 15.5*111.4 ±2.8116±13.3t

3.30 ± 0.245.33 ± 0.24*

2.85 ± 0.303.91 ± 0.39U

Data are microunits of insulin released per islet per hour and picomoles of ATP per islet. They represent the means ± SE of 10-28observations from three to five experiments. *P< 0.001, tP < 0.05 compared with 2 mmol/1 glucose; fP < 0.001, §P < 0.01 comparedwith control at the same incubation condition.

malizing the data to account for the absolute magnitude ofcurrent demonstrates, however, that the glucose sensitivityof the K jp channel is actually very similar in the two strainsof rat. The increased density of K^p current in the GK ratsexplains why in the absence of glucose they have a more neg-ative resting potential than observed in controls. The mem-brane potential and electrical activity, hence calcium entry andinsulin secretion, of the (3-cell is exquisitely controlled bythe level of K^p channel activity (current) (20,27,39,41). Thepresence of larger KATP currents encountered in p-cells fromGK rats suggests that for a given glucose concentration, theirmembrane potential will be more hyperpolarized and thus lesselectrically active than in normal subjects. Indeed, thisexpectation is supported by the membrane potential in 5mmol/1 glucose being significantly more hyperpolarized in (B-cells from GK rats than from control subjects. A similarmechanism may account for the lower secretion measuredwith higher glucose concentrations in the intact islet.

This difference in K^p current density cannot, however,totally account for the lower insulin secretion observed in GKrats because under the experimental conditions, where K^pchannels were held open and the effects of glucose wereindependent of their closure, insulin release was still signifi-cantly lower in GK rat islets than in controls. This finding sug-gest that defects in the secretory pathway are also to befound distal to KATP channel closure. Since Ca2+-inducedinsulin release measured in both single (3-cells and in per-meabilized islets from GK rats is not impaired (10,11), it is pos-sible that the decrease in insulin release from GK rat isletsmay result from diminished Ca2+ entry into the p-cell. Thiswould result in a lower elevation of intracellular calciumthan normally occurs, contributing to the impairment ininsulin secretion. Kato et al. (37) recently demonstrated thatthe amplitude of Ca2+ currents in (3-cells from GK rats werein fact larger than those measured from normal rats. In thisstudy, we found that there are no significant differencesbetween the properties of Ca2+ currents we measured in (3-cells from normal and GK rats. Moreover, both the magnitudeof the Ca2+ currents and the relative contributions from T- andL-type Ca2+ channels were almost identical between the twostrains of rat. It is not possible to directly compare our Ca2+

currents with those of Kato et al. (37) because they used a high(30 mmol/1) concentration of Ba2+ and different pipette solu-tions to measure inward currents, both of which will dra-matically alter the properties of the currents recorded (21).Nevertheless, our demonstration that little difference existsin Ca2+ currents between (3-cells from control and diabetic rats

measured at physiological Ca2+ and glucose concentrationssuggests that Ca2+ current dysfunction is unlikely to underliethe impaired insulin secretion in GK rats.

We did not investigate the modulation of L-type Ca2+ cur-rents by glucose, like that demonstrated in (3-cells from mice(22). However, the glucose concentration we used (5.5mmol/1) produces a rate of metabolism close to maximal (thisstudy). Kato et al. (37) suggested that glucose modulation ofthe Ca2+ current is impaired in p-cells from the GK rat. How-ever, they recorded Ca2+ currents of similar magnitude tothat in controls at high glucose concentrations, levels atwhich insulin release is substantially reduced. Both our dataand that of Kato et al. (37) therefore suggest that differencesin Ca2+ current do not contribute to the impaired insulinrelease observed in GK rats.

Given that (3-cells from both normal and GK rats have a sim-ilar exocytotic response to Ca2+ influx at the level of the sin-gle cell, at least within a period of 3 min from the beginningof whole-cell recording, suggests that the early events in theCa2+ entry secretion pathway are essentially normal in the GKrats. Although we show that the insulin content is strikinglysimilar in islets from the two species of rat, it is possible thatsecretory vesicles from the GK rat may contain less insulinthan in normal rats; therefore, less insulin may be expectedto be released for a given amount of exocytosis. However, thisidea is not supported by the demonstration that both normaland GK rat (3-cells had comparable insulin secretoryresponses when stimulated with a fixed level of calcium andATP in studies using permeabilized islets (10,11), data that cor-roborate our own observations. Finally, our capacitanceexperiments also indicate that endocytosis of insulin secre-tory granules in GK rat can occur and superficially appearsto be normal.

An important metabolite that is thought to couple glucosemetabolism to K^p closure and membrane depolarization inthe pancreatic (3-cell is ATP (38,39). It has previously beenreported that ATP levels are similar in control and GK rat isletswhen incubated at stimulatory glucose concentrations (40).In the present study, we have found that although incubationin the presence of 10 mmol/1 glucose increased the ATP con-tent of GK rat islets, this increase was smaller than thatobserved in control islets and may explain the slower rate ofblock of K ^ current produced by glucose we observed in sin-gle p-cells from GK rats in 5 mmol/1 glucose.

It has previously been demonstrated that glucose can pro-mote insulin release under conditions in which K^p channelsare held open by diazoxide and the membrane depolarized


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STUDIES OF SINGLE li-CELLS FROM GK RATS

with 40 mmol/1 K+ (41). The mechanism underlying thisaction of glucose and other nutrient secretagogues has notbeen established although it may involve paracrine interac-tions or a direct effect of metabolites, such as ATP, on thesecretory machinery of the (3-cell (42). Neither the capacitancenor permeabilized islet studies, however, address the questionof whether a difference exists in the relationship between ATP,or another metabolite, and secretion in the two strains of rat.Such a difference may underlie, at least in part, the impairedinsulin secretion in this diabetic animal model. Furthermore,if such a difference does indeed exist, any effect on insulinsecretion in (3-cells from the GK rat may be exacerbated bytheir reduced ATP content. A dysfunction in such mecha-nism^) may therefore explain why glucose-stimulatedinsulin secretion in islets of GK rats is still markedly lowerthan in controls, particularly when one considers that secre-tion induced by calcium at a fixed level of intracellular ATPin both single p-cells and islets (over short and longtimescales, respectively) is similar in GK and control rats.

One original aim of our paper was to determine if our GKrats were diabetic because of dysfunctional ion channelbehavior like that described in other GK rat colonies. As wehave shown, this notion does not appear to be the case withour colony. It is important to check whether this is true forother GK rat colonies. In conclusion, although our findingssuggest that the impaired insulin secretion in islets from GKrats may, in part, result from a failure to close an adequatenumber of K^p channels to bring about membrane depolar-ization, dysfunction also occurs in events distal to K^p clo-sure. This does not, however, arise from an impairment in thecalcium secretion coupling but may reside in factors thatcouple metabolism to secretion.

ACKNOWLEDGMENTSM.F. was a Rhodes Scholar. P.A.S. was a recipient of WellcomeCareer Development Award 042345.

We thank the British Diabetic Association and the WellcomeTrust for their support.

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Electrophysiological and Metabolic Characterization of - Diabetes

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