Potassium currents in ventricular myocytes from genetically diabetic rats

Katsuharu Tsuchida and Hiroshi Watajima

Research Center, Taisho Pharmaceutical Company, Ohmiya, Saitama 330, Japan

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

Our previous study demonstrated the longer duration of action potential in ventricular myocytes from genetically diabetic WBN/Kob rats without change in calcium channel density compared with age-matched controls [Tsuchida, K., H. Watajima, and S. Otamo. Am. J. Physiol. 267 (Heart Circ. Physiol. 36): H2280-H2289, 1994]. In the present study we examined the alteration of potassium currents, especially transient outward current, in ventricular myocytes of genetically diabetic WBN/Kob rats. WBN/Kob rats gradually develop hyperglycemia with aging and show some similarity to non-insulin-dependent diabetes mellitus models, which differ from the insulin-dependent streptozotocin-treated rat model. The density of the intracellular calcium ion-independent transient outward current (Ito) from 17- to 19-mo diabetic rat myocytes was significantly smaller than that from age-matched control rat myocytes. In addition, the density of Ito from 17- to 19-mo rat myocytes was significantly less than that from 2-mo rat myocytes, suggesting that aging-induced alteration of Ito was accelerated by the diabetic state. The steady-state inactivation curves of Ito, the recovery from Ito inactivation, and the other outward currents were not significantly altered between diabetic myocytes and age-matched control myocytes. In conclusion, the prolonged duration of action potential from genetically diabetic rat myocytes is mainly due to the depressed Ito.

cardiomyocytes; transient outward current

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

IT HAS LONG BEEN KNOWN that patients with diabetes mellitus have increased risk of mortality from cardiac failure that cannot simply be explained in terms of atherosclerosis, hyperlipidemia, or hypertension. Many studies have been performed to investigate the cardiac function of diabetic animals, and a variety of mechanical, electrophysiological, and biochemical abnormalities have been identified in the myocardium of diabetic animal models (7). Chemical-induced diabetic models, especially the streptozotocin (STZ)-induced diabetic model, are frequently used as a model for insulin-dependent diabetes mellitus (IDDM) to investigate these abnormalities. With use of this animal model, contractile abnormalities have been reported (7), and this dysfunction has been related to biochemical changes such as the altered distribution of myosin isozymes (4); a decrease in uptake of Ca2+ into sarcoplasmic reticulum (6) and sarcolemma (9); decreased activity of Na+-K+-adenosinetriphosphatase (14), Na+-H+ exchange (15), and Na+-Ca2+ exchange (16) in the sarcolemmal membrane; and Ca2+ binding capacity (20). Abnormalities of electrophysiological phenomena have also been demonstrated in STZ-induced diabetic rats. Ventricular and atrial action potentials were prolonged when the measurement was done for a relatively longer term after STZ injection (1, 15). The L-type calcium current was not altered from 6 days to 2 mo (12, 17) but was decreased from 5 to 7 mo (28), whereas the transient outward potassium current was decreased significantly from 6 days to 7 mo after STZ injection (12, 17, 24, 28). Our previous study using genetically diabetic rats (WBN/Kob) demonstrated that the ventricular action potential duration is prolonged (25). STZ-treated rats closely resemble the IDDM model, except in a special case (23), whereas WBN/Kob rats show some similarities with the non-insulin-dependent diabetes mellitus (NIDDM) model. That is, male WBN/Kob rats have been reported to develop hyperglycemia, glycosuria, polyuria, and glucose tolerance from ~9 mo of age, with a gradual and moderate decrease in serum insulin levels (19). The rats survive without the administration of insulin, in contrast to Bio-Breeding rats, a genetic model of IDDM. The pathological process progresses slowly in WBN/Kob rats. Much clinical attention has been paid to NIDDM-induced cardiomyopathy (7, 23). We have previously reported that the L-type Ca2+ channel did not alter significantly, although the response of the L-type Ca2+ channel to beta -adrenergic stimulation decreased in WBN/Kob rats (25). In this study, we first examined changes in the potassium currents, especially the transient outward potassium current, in this strain of genetically diabetic rats.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

Animal model of diabetes. A particular strain of Wistar-derived WBN rats has been maintained at the Institute of Pathology, Bonn University (Bonn, Germany). Several of these animals were brought to Japan by Dr. O. Kobori in 1976 and became known as WBN/Kob rats. A colony of these rats is currently being maintained at the Shizuoka Laboratory Animal Center (Shizuoka, Japan). Male WBN/Kob rats of 17 to 19 mo of age and age-matched Wistar rats (purchased from the Shizuoka Laboratory Animal Center) were used. The main clinical sign of diabetes, glucosuria, was evidently detected at ~14 mo of age, after a marked glucose intolerance at 12 mo. Thereafter, some animals developed hyperlipidemia and gradual emaciation (19, 26). After purchase at 6 mo and until they were used in the experiment, all rats were maintained at the Taisho Pharmaceutical Animal Laboratory and were fed standard rat chow.

Cell preparation. Single ventricular cells were isolated according to the methods previously reported (25). Briefly, the rat was anesthetized with pentobarbital sodium (50 mg/kg ip), and the heart was rapidly excised and attached to a Langendorff perfusion apparatus. The heart was then retrogradely perfused for 2-3 min with nominally calcium-free Krebs-Henseleit solution equilibrated with 95% O2-5% CO2 at 36°C. The Krebs-Henseleit solution contained (in mM) 130 NaCl, 4.8 KCl, 1.2 MgSO4, 1.1 NaH2PO4, 25 NaHCO3, and 12.5 glucose. Enzymatic digestion was achieved by recirculating the perfusion apparatus with the calcium-free Krebs-Henseleit solution containing 40-170 U/ml collagenase (Yakult, Tokyo, Japan). The perfusion pressure was maintained at ~80 mmHg. Enzymatic perfusion was stopped after 15-30 min. The heart was then washed with Kraftbrühe (KB) solution containing (in mM) 70 L-glutamic acid, 5 KCl, 20 taurine, 5 KH2PO4, 11 glucose, 5 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and 0.5 ethylene glycol-bis (beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) free acid. The ventricle was separated from the whole heart and minced into small pieces. Cells were filtered and stored in KB solution at 4°C before being used in the electrophysiological experiment. After storage for 3-6 h in KB solution, the cells were taken into the experimental chamber, which was perfused with Ca2+-containing Tyrode solution when the electrophysiological experiment was performed.

Electrophysiological recording. Some cells were transferred to a recording chamber (0.3 ml vol) placed on the stage of an inverted microscope (Diaphot TMD, Nikon, Tokyo, Japan), and the chamber was perfused at a constant rate of 1-2 ml/min. Membrane currents were recorded using the whole cell patch-clamp method described by Hamill et al. (8) by use of a patch-clamp amplifier (CEZ-2300, Nihon Kohden) connected to pClamp software program (Axon Instruments, Burlingame, CA) or an Atari Mega ST4-operated EPC-9 patch-clamp system (Heka, Lambrecht, Germany). Patch pipettes (2-4 MOmega ) were fabricated using a puller (PP-83, Narishige, Tokyo) and were heat polished (MF-83, Narishige). To record transient outward current (Ito), we filled the pipettes with solution containing (in mM) 140 KCl, 2 MgCl2, 1 CaCl2, 10 HEPES, 11 EGTA, and 5 Na2ATP (pH ajusted to 7.2 with KOH). The extracellular mediums were standard Tyrode solution containing Co2+, to eliminate a calcium ion current (ICa), or a Na+-free Co2+-containing solution to eliminate the tetrodotoxin (TTX)-sensitive Na+ current, the Na+-activated K+ current, and ICa. The composition of Na+-free solution was (in mM) 135 choline chloride, 5.4 KCl, 1 MgCl2, 1.8 CaCl2, 10 HEPES, 10 glucose, and 3 CoCl2 (pH 7.4 with KOH). In some experiments, 4-aminopyridine (4-AP; Sigma Chemical, St. Louis, MO) was used to block Ito, and TTX (Sankyo, Tokyo, Japan) was used to block sodium ion current (INa).


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Fig. 1.   Representative outward currents recorded in single ventricular myocytes isolated from diabetic (17-19 mo) and age-matched control rat heart and in young adult (2 mo) normal rat ventricular myocytes. Currents were elicited by applying a 300-ms depolarizing step in 10-mV increments to +70 mV from a holding potential of -70 mV every 5 s. Effects of 5 mM 4-aminopyridine (4-AP) on the current elicited by a test pulse to +70 mV from -70 mV are also represented. Six families of current tracings are from different myocytes. Total cell capacitances (pF) are as follows: right (R) 170, left (L) 132 in 17- to 19-mo control rats; R 153, L 186 in 17- to 19-mo diabetic rats; R 117, L 144 in 2-mo rats.

Concerning the determination of cell membrane capacitance, the procedure described below was used. Just after the patch was broken, pulses with a duration of 50 ms, from -60 to -62 mV, were applied to the cell. The exponential components of the decaying current were determined. Two time constants were obtained corresponding to the electrode and membrane capacitance. The time constant of the electrode was <0.02 ms, and the time constant of the membrane capacitance was just under 2 ms. After the electrode capacitance was compensated for, the capacitance of the membrane was calculated according to the equation
C<SUB>m</SUB> = <IT>T</IT><SUB>c</SUB> ⋅ <IT>I</IT><SUB>o</SUB>/<IT>E</IT><SUB>m</SUB>[1 − (<IT>I</IT>/<IT>I</IT><SUB>o</SUB>)]
where Cm is the membrane capacitance, Tc is the time constant of the membrane capacitance, Io is the maximum capacitance current value, Em is the amplitude of the voltage step, and I is the steady-state current. The series resistance (Rs) was calulated as
<IT>R</IT><SUB>s</SUB> = <IT>E</IT><SUB>m</SUB>/<IT>I</IT><SUB>o</SUB>
which ranged from ~4 to 10 MOmega . In some cases, membrane capacitance and series resistance were determined electronically. Then membrane capacitance was compensated for, and series resistance was reduced maximally for the voltage-clamp experiments to examine the membrane currents.

Data analysis. Statistical significance was determined with the Student's t-test for unpaired data or with the repeated-measures analysis of variance test for multiple comparison. A value of P < 0.05 was considered significant.

    RESULTS AND DISCUSSION
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

Plasma glucose level and clinical state. Plasma glucose levels were 558 ± 16 (SE) mg/dl with 17- to 19-mo WBN/Kob rats (n = 34), whereas plasma glucose levels of age-matched control Wistar rats were within the range of ~110-140 mg/dl. Almost all WBN/Kob rats showed glucosuria and ruffled hair including transient alopecia and seemed sluggish at 17-19 mo.

Effects of genetic diabetes on Ito. Outward currents were recorded after blocking ICa with 3 mM Co2+ or INa with 30-50 mM TTX, or changing extracellular NaCl with choline chloride and the intracellular Ca2+-activated current with 11 mM EGTA added in the pipettes. From a holding potential of -70 mV, depolarizing pulses in 10-mV increments were applied from -60 mV up to +70 mV. Ito was elicited by depolarizing pulses more positive than -20 mV in both normal and diabetic ventricular myocytes. Application of 5 mM 4-AP suppressed Ito, and the 4-AP-insensitive component of the outward current remained. Figure 1 shows typical currents obtained from diabetic (17-19 mo), age-matched control (17-19 mo), and young (2-mo) rat ventricular myocytes.

Ito consists of 4-AP-sensitive and -insensitive components. The 4-AP-insensitive Ito is reported to be activated by increased intracellular calcium concentration ([Ca2+]i). Because we used a pipette solution containing a relatively high concentration of EGTA (11 mM), the [Ca2+]i-dependent activation of Ito could be ruled out, thereby leaving only one component of Ito, namely, the 4-AP-sensitive component shown in Fig. 1. Hereafter, we will refer only to this component of Ito. The amplitude of Ito was expressed as the difference in current between the peak current amplitude and the terminal component of the current at the end of depolarizing test pulses of 300-ms duration. The amplitude of Ito is shown in Fig. 2A. The current density of the difference in current between peak and 300-ms terminal currents was significantly less in diabetic ventricular myocytes than in the age-matched control myocytes. Apkon and Nerbonne (2) described a slowly activating and inactivating K+ current (IK in their terminology) in rat ventricular myocytes, which was sensitive to external tetraethylammonium but not to 4-AP. Thus the terminal component at the end of 300-ms pulses is considered to consist of two different currents. One is the residual component of the slowly inactivating Ito at the end of 300-ms pulses, and the other is the 4-AP-insensitive outward current. To determine the total Ito as the 4-AP sensitive current, the difference in current between the absence and the presence of 5 mM 4-AP was obtained and shown in the inset of Fig. 2A. The density of the 4-AP-sensitive current was also significantly less in the diabetic ventricular myocytes than in the age-matched control myocytes. The decrease of Ito density in the genetically diabetic WBN/Kob rats is consistent with findings reported in STZ-treated diabetic rats (12, 17, 28). We used aged rats of 17-19 mo as controls, because WBN/Kob rats gradually develop hyperglycemia with aging. We examined the influence of aging on Ito as well. Wei et al. (29) demonstrated that the action potential duration in the senescent (24-mo) rat ventricular myocytes was longer than that in the young adult (7-mo) rat myocytes. Other authors have reported similar changes of action potential duration (3). Walker et al. (27) demonstrated that Ito was depressed more markedly in the cardiac cells of the aged (24- to 25-mo) rat than in those of young adult (2- to 3-mo) rats. The current density of Ito from 17- to 19-mo rat myocytes was significantly less than that from 2-mo rat myocytes (Fig. 2A). The decay process of Ito seemed to be fitted with biexponential curves with fast and slow time constants (tau f and tau s, respectively). Because the pulse duration was too short to determine tau s correctly, we have presented only tau f. At +70 mV, tau f was 32.8 ± 2.9 ms (n = 21) in 17- to 19-mo control, 32.3 ± 1.2 ms (n = 19) in 17- to 19-mo diabetic, and 33.4 ± 1.5 ms (n = 14) in 2-mo normal rats. All values were not significantly different. Possible values of tau s seemed roughly ten times longer than those of tau f. The terminal current amplitude at the end of 300-ms pulses is shown in Fig. 2B. The terminal component was not altered in WBN/Kob rat myocytes compared with that in the age-matched control rat myocytes. Jourdon and Feuvray (12) and Wang et al. (28) described the significant decrease in this time-independent outward current in the STZ-treated rat myocytes. The present result is different from their results. In addition, the current amplitude of the terminal component was not altered between examinations of 17- to 19-mo rats and 2-mo rat myocytes (Fig. 2B).


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Fig. 2.   Current-voltage relationships of transient outward current (Ito) and terminal components of the outward current in ventricular myocytes from diabetic (17-19 mo) and age-matched control rats, and from young adult (2 mo) rats. Amplitude of Ito was measured as the difference in current amplitude between the peak current amplitude and the terminal current amplitude at the end of 300-ms test pulses. A: Ito; B: terminal current. bullet , Diabetic myocytes (n = 23); open circle , age-matched control myocytes (n = 22); square , young adult myocytes (n = 14). Inset a: voltage-clamp protocol; inset b: 4-AP-sensitive current induced by a 300-ms test pulse to +70 mV from -70 mV. Control, n = 16; WBN/Kob, n = 16. Values are means ± SE. * P < 0.05 vs. age-matched control (17-19 mo) for diabetic rat myocytes, and + P < 0.05 vs. young adult (2 mo) rat for aged (17 - 19 mo) rat myocytes at each membrane potential (Student's t-test). A supplemental explanation: repeated-measures analysis of variance test showed that current (Ito)-voltage relationships were significantly (P < 0.05) different between diabetic rats and age-matched control rats. Current (Ito)-voltage relationships were also significantly (P < 0.05) different between 2-mo rats and 17- to 19-mo rats.

Modification of inactivation kinetics of Ito. In this series of experiments, Ito was also expressed as the change in current between the peak amplitude and the terminal component of the current. The steady-state inactivation kinetics of Ito were compared between the diabetic myocytes and the age-matched control myocytes. A test pulse to +70 mV (duration 300 ms) was preceded by 1-s conditioning prepulses to various potentials (from -80 mV to 0 mV). The relative amplitude of Ito (normalized by taking the value at -80 mV as unity) was plotted against conditioning potentials, and the data were fitted by the following Boltzmann distribution function with a least squares method
<IT>I</IT>/<IT>I</IT><SUB>max</SUB> = 1/[1 + <IT>e</IT><SUP>(<IT>V</IT><SUB>m</SUB>−<IT>V</IT><SUB>0.5</SUB>)/<IT>k</IT></SUP>]
where I/Imax is the relative amplitude of Ito, Vm is the conditioning voltage, V0.5 is the voltage of half-inactivation, and k is the slope factor. The values of V0.5 and k were -43.9 ± 2.1 and 7.7 ± 0.54 mV for age-matched control myocytes, and -47.3 ± 3.1 and 6.9 ± 0.38 mV for diabetic myocytes, respectively (Fig. 3). The value of V0.5 was not altered significantly between the two groups. The value of k was not significantly different between the two groups, either. Furthermore, the values of V0.5 and k were -51.6 ± 2.9 and 6.6 ± 0.65 mV for 2-mo rat myocytes. The V0.5 value in 2-mo rat myocytes shifted several mV negatively, and significantly, compared with 17- to 19-mo normal rat myocytes, without any change in k.


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Fig. 3.   Steady-state inactivation curves of Ito in diabetic (17-19 mo) and age-matched controls and young adult (2 mo) rat myocytes. I/Imax, relative amplitude of Ito. bullet , Diabetic (n = 18) myocytes; open circle , controls (n = 16); square , young adult myocytes (n = 10). Values are means ± SE. Inset: voltage-clamp protocol.

The time course of recovery from inactivation of Ito was also compared between the diabetic and age-matched control rat myocytes by use of a double-pulse method. Double pulses to +70 mV from a holding potential of -70 mV (each pulse duration 300 ms) were applied every 10 s, while the interpulse interval was increased from 10 ms to 1 s. The relative amplitude of Ito induced by the second pulse vs. the Ito amplitude induced by the first pulse was plotted against the interpulse interval. Recovery from its inactivation was not different between diabetic myocytes and age-matched control rat myocytes, as shown in Fig. 4. In addition, the recovery of Ito was not altered in normal 17- to 19-mo rat myocytes compared with 2-mo rat myocytes. The previous studies showed that the kinetics of steady-state inactivation and recovery kinetics from inactivation were not altered in STZ-treated rats with short treatment periods of 1.5-2 mo (12) but were slowed in long-term (5- to 7-mo) STZ-treated rats (28). It seems that the kinetic changes of Ito may occur in the severely diseased myocardium with long-term STZ-induced diabetes.


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Fig. 4.   Time course of recovery from inactivation of Ito in ventricular myocytes from diabetic (n = 18), age-matched (17-19 mo) controls (n = 17), and young adult (2 mo) myocytes (n = 12). Double pulses (each duration 300 ms) from -70 mV to +70 mV were applied at varying interpulse intervals (from 10 ms to 1 s) every 10 s. bullet , Diabetic myocytes; open circle , age-matched control myocytes; square , young adult myocytes. Values are means ± SE. Inset: voltage-clamp protocol.

Effects of diabetes on inward rectifying current. The current amplitudes, elicited by hyper- and depolarizing steps from -70 mV to up to -10 to about -110 mV, were not different between diabetic rat myocytes and age-matched control rat myocytes (Fig. 5). Furthermore, the terminal component of the outward current elicited by depolarizing test pulses up to ~0 mV from a holding potential of -70 mV was not altered in diabetic rat myocytes compared with age-matched control myocytes (Fig. 2B). These results suggest that the inward rectifying potassium current (IK1) was not altered in the diabetic state. No alteration in the IK1 has also been demonstrated by other authors in STZ-treated diabetic rats (12, 17, 20), whereas reduced IK1 was reported in other diseased cardiac myocytes (13).


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Fig. 5.   Inward rectifying potassium current density in ventricular myocytes from diabetic and age-matched (17-19 mo) control and young adult (2 mo) rats. Steady-state inward current measured at end of 1-s duration of hyperpolarizing test pulses at 10 mV increased voltage steps from a holding potential of -70 mV to -10 up to -110 mV. bullet , Diabetic (n = 15); open circle , age-matched (17-19 mo) control (n = 13); square , young adult (2 mo) (n = 10) myocytes. Values are means ± SE. Inset: voltage-clamp protocol.

Summary of results and significance of the decrease in Ito. The present study has, first of all, demonstrated that the current density of Ito in ventricular myocytes isolated from genetically diabetic rats sharing the characteristics with NIDDM was significantly less than that from 17- to 19-mo-old matched control rats. The current density of Ito from aged (17- to 19-mo) rats was significantly less than that from young adult (2-mo) rats. Thus the Ito density was reduced by aging, and the reduction was further accelerated by a diabetic state. Ito is considered to be one of the most important repolarizing currents in rat myocytes (11). The present study indicates that the lower Ito density, but not kinetic changes of the Ito, may be responsible for the longer duration of action potential in WBN/Kob rats, as already shown in our previous study (25). Shimoni et al. (24) demonstrated that the diabetic state exerted differential effects on the Ito in epicardial and endocardial myocytes from the left ventricle of short-term (6- to 7-day) STZ-treated rats. Such regional differences were not a focus of the present study but may be important in understanding the pathophysiology of the diabetic heart more thoroughly. We previously demonstrated that the L-type Ca2+ current was not altered in 19-mo WBN/Kob rat ventricular myocytes in vitro under the condition of the inhibition of the potassium current by use of the whole-cell patch-clamp technique (25). However, the in situ ventricular myocytes exist in circumstances without any artificial restriction of the potassium current. The action potential duration of rat ventricular cells is short enough to allow maximum Ca2+ influx from extracellular medium, so that the lengthening of the action potential duration due to the inhibition of Ito may lead to increased Ca2+ influx and subsequent enhancement of Ca2+ release from intracellular stores, resulting in some compensatory effects on the decreased contractile force of diabetic myocardium (10, 21). Our previous study (25) indicated that WBN/Kob rats demonstrated decreased contractile force in situ. Thus the compensation resulting from lengthening the action potential duration was not considered to be sufficient for many reasons (see the introductory section of this paper) other than the decreased ICa being responsible for the decreased contractile force in WBN/Kob rats.

Recently, Xu et al. (30) demonstrated that the decreased Ito density may be caused by the decrease in cellular glucose metabolism in ventricular myocytes of short-term (14 days to 1 mo) STZ-treated rats (30). Such biochemical changes may be involved in downregulating Ito in genetic NIDDM myocytes. The lower density of the Ito observed in the diabetic ventricular myocytes may result from a decrease in the number of channels. Concerning a molecular basis of the decreased Ito in diabetic myocytes, because Dixon et al. (5) demonstrated that both the ventricular potassium (Kv) 4.2 and Kv 4.3 channels were likely to contribute to the Ito in rat heart, the alteration of the expression of these genes may account for the altered Ito density in WBN/Kob rats. The molecular alterations in the channel proteins remain to be elucidated as an interesting problem.

    FOOTNOTES

Address for reprint requests: K. Tsuchida, Research Center, Taisho Pharmaceutical Co., Ltd., 1-403 Yoshino-cho, Ohmiya, Saitama 330, Japan.

Received 25 February 1997; accepted in final form 4 June 1997.

    REFERENCES
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

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AJP Endocrinol Metab 273(4):E695-E700
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