(Circulation. 1995;91:764-770.)
© 1995 American Heart Association, Inc.
Articles |
Induction of Plasminogen Activator Inhibitor Type-1 (PAI-1) by Proinsulin and Insulin In Vivo
From the Cardiovascular Division, Washington University School of Medicine, St Louis, Mo, and the Department of Medicine, The University of Vermont College of Medicine, Burlington.
Correspondence to Burton E. Sobel, MD, Department of Medicine, Medical Center Hospital of Vermont, Fletcher House 311, Burlington, VT 05401.
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Background Fasting hyperinsulinemia (reflected by elevations in immunoreactive "insulin") is typical of patients with non–insulin-dependent diabetes mellitus (NIDDM) and is often associated with obesity and hypertension. The elevated concentrations detected are indicative not only of insulin but also of its immunologically cross-reactive precursors, including proinsulin. Fasting hyperinsulinemia appears to be associated with decreased fibrinolytic activity in blood, which results from increased activity of plasminogen activator inhibitor type-1 (PAI-1), a potential independent risk factor for coronary artery disease. Patients who were given proinsulin in a previous clinical study by others exhibited an increased incidence of cardiovascular events. Thus, a "proinsulin–PAI-1 axis" may predispose to coronary thrombosis. To define the possible presence of such an axis, this study was designed to determine whether insulin, its precursors, or both increase the concentrations of PAI-1 in rabbits in vivo.
Methods and Results Equimolar proinsulin (n=10), insulin (n=11), C-peptide (n=4), or vehicle alone (n=10) was administered intravenously over 1 hour to euglycemic, conscious rabbits. Plasma PAI-1 activity increased 3.8-fold with proinsulin (P=.002) and 3.6-fold with insulin (P=.002). By contrast, no increase occurred after C-peptide or vehicle was administered. The increased PAI-1 activity was shown to be attributable to PAI-1 protein by reverse fibrin autography. As judged from changes in mRNA in tissues, proinsulin and insulin increased PAI-1 gene expression within 3 hours by 2.1- and 2.1-fold, respectively, in aorta (P=.025 each) and by 1.9- and 2.4-fold in liver (P=.015 and P=.001), with return of values to baseline within 24 hours (n=4 experiments in each case).
Conclusions These results extend our previous observations from studies in vitro and suggest that hyperinsulinemia attributable to augmented concentrations of proinsulin and insulin in plasma increase plasma PAI-1 activity and may contribute to acceleration of atherosclerosis and impairment of coronary thrombolysis in patients with NIDDM.
Key Words: insulin • diabetes mellitus • fibrinolysis
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Introduction |
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Both the incidence and severity of coronary artery disease increase in patients with non–insulin-dependent (type II) diabetes mellitus (NIDDM). Fasting hyperinsulinemia (increased immunoreactive "insulin") is typical and has been implicated as a risk factor for coronary disease.1 Conventional radioimmunoassays for insulin exhibit cross-reactivity with precursors of insulin, including proinsulin des31,32proinsulin, and des64,65proinsulin.2 In fact, proinsulin and proinsulin-like molecules account for up to 60% of total immunoreactive insulin in the plasma of patients with NIDDM, compared with 10% to 20% in that of nondiabetic subjects.2 3 For reasons that have not yet been clarified, in a clinical trial by others,4 diabetic patients treated with proinsulin for at least 1 year exhibited cardiovascular events 7 to 18 times more frequently than those treated with insulin. One factor that may be responsible for these untoward events is reduced plasma fibrinolytic activity secondary to increased concentrations of plasminogen activator inhibitor type-1 (PAI-1), the major physiological inhibitor of endogenous fibrinolysis in blood.5 6
Reduced fibrinolysis may predispose patients to deposition of intramural and intraluminal fibrin potentiating microthrombotic or macrothrombotic occlusions in atherosclerotic coronary arteries. Incorporation of fibrin in the vessel wall may exacerbate atherogenesis, as judged from the robust accumulation of fibrin in human atheroma7 and from the stimulation by fibrin of vascular smooth muscle cell migration8 and of accumulation of LDL and especially lipoprotein (a).9
We and others have found that proinsulin, proinsulin split products, and insulin stimulate the synthesis of PAI-1 in Hep G2 (highly differentiated human hepatoma) cells10 11 12 and in human hepatocytes in primary culture.13 Furthermore, we found that glucose, in concentrations seen in plasma in hyperglycemic patients, induces synthesis of PAI-1 in aortic endothelial cells in vitro.14 Accordingly, the present study was performed to determine whether infusions of proinsulin in vivo can induce changes in plasma PAI-1 activity that may account for reduced fibrinolytic activity in blood in patients with NIDDM.
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Methods |
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Materials
Recombinant human insulin (26 insulin units/mg) was purchased from Sigma Chemical Co, dissolved at a concentration of 10 mg/mL in sterile saline (0.9% sodium chloride, Abbott) supplemented with 1% (vol/vol) glacial acetic acid, and diluted in saline supplemented with 0.5% albumin (bovine, fraction V, low endotoxin; Sigma) to minimize the adherence of insulin to storage devices and infusion systems. Human proinsulin was provided by Eli Lilly, and synthetic human C-peptide was purchased from Sigma. Both were dissolved in saline with 0.5% albumin. Protein concentrations were verified by conventional assay with bicinchoninic acid (Pierce). Glucose (Sigma) was prepared as a 20% (wt/vol) sterile solution in saline.
Contamination with endotoxin was excluded in all reagents and vehicles by testing with Limulus amebocyte lysate (Pyrotell assay, Associates of Cape Cod; sensitivity for detection of endotoxin, 0.0125 ng/mL).
Procedures in Rabbits
Preparations
Care and
handling of rabbits conformed with the Washington
University Committee on Human Care of Laboratory Animals and
Declaration of Helsinki standards. New Zealand White rabbits (3.7±0.4
kg body wt, mean±SD) were obtained from Doe Valley Farms (Bentonville,
Ark). Experimental procedures were implemented in conscious animals
tranquilized with injection of 0.5 to 0.75 mL IM acepromazine (10 mg/mL
PromAce, Aveco). The rabbits were assigned randomly to four treatment
groups. Infusions of insulin, proinsulin, C-peptide, or vehicle alone
(10 mL each) were administered by marginal ear vein catheter over 1
hour with a syringe infusion pump (No. 22, Harvard Apparatus). Doses
were (µg/kg, equimolar) insulin 37, proinsulin 61, and C-peptide 23.
The insulin concentration (1 U/kg) was selected to yield plasma
concentrations in the upper range of those in patients with NIDDM.
Euglycemic Clamp Technique
To maintain
concentrations of blood glucose in the normoglycemic
range for rabbits (110 to 150 mg/dL),15 glucose was
monitored every 10 minutes with Dextrostix reagent strips (Miles) and a
reflectance meter. Glucose was infused through a separate marginal
ear-vein catheter to maintain euglycemia (Fig 1
).
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3
hours). The glucose infusion rate was adjusted to maintain blood
glucose in the physiological range for rabbits (110 to 150
mg/dL).
Acquisition of Plasma and Tissue Samples
To obtain
plasma, whole blood was collected in plastic syringes
from a central ear artery catheter with a two-syringe technique and
transferred immediately into tubes containing sodium citrate (12.9
mmol/L final concentration). The sample volume (2 mL) was replaced by
an equal volume of saline. Plasma was separated by centrifugation at
1000g at 4°C for 10 minutes, frozen in liquid nitrogen,
and stored at -20°C until assay.
To obtain tissue samples, rabbits were given an overdose of xylazine intravenously (AnaSed, Lloyd Laboratories) at selected intervals. Heart, aorta, and liver were removed rapidly and rinsed twice in cold PBS. Representative tissue samples were snap-frozen in liquid nitrogen and stored at -70°C until assay.
PAI-1 Activity in Plasma
PAI-1 activity was assayed
spectrophotometrically. Samples were
incubated with exogenous tissue-type plasminogen activator (TPA)
(KabiVitrum) at room temperature for 10 minutes in conditions under
which PAI-1 but not other low-affinity inhibitors could bind to the
TPA. Subsequently, the samples were acidified and snap-frozen to
eliminate residual 
2-antiplasmin activity. Residual TPA
activity was assayed spectrophotometrically by incubation with
plasminogen (Kabi) and a chromogenic substrate, S-2251
(KabiVitrum).16 Standard curves were obtained with serial
dilutions of pooled normal rabbit plasma. Results were expressed as
arbitrary units (AU), where 1 AU is defined as the amount of PAI-1 that
inhibits 1 IU of TPA within 10 minutes.
Reverse Fibrin Autography
Plasma samples for reverse fibrin
autography were subjected to
SDS-PAGE on stacking gels of 4% and separating gels of 7.5%
acrylamide either directly or after immunoprecipitation of PAI-1. For
immunoprecipitation of PAI-1, rabbit plasma was diluted 1:4 with PBS
and incubated with goat anti-human PAI-1 antibody (No. 395G, an
antibody that cross-reacts with rabbit PAI-1; American Diagnostica) at
a final concentration of 20 µg/mL with gentle agitation at 4°C
overnight. Subsequently, the mixture was incubated with
Sepharose-linked protein G (Pharmacia LKB) at room temperature for 2
hours. Antibody-bound PAI-1 was pelleted by centrifugation in a
microcentrifuge, and the pellets were washed in PBS with 0.1% SDS,
0.5% Nonidet P-40, and 0.1% deoxycholic acid twice and in PBS alone
once, resuspended in reducing buffer, and heated at 100°C for 3
minutes. The supernatant fractions containing concentrated rabbit PAI-1
were then subjected to SDS-PAGE.
Reverse fibrin autography was performed with a modification of the procedures described previously17 18 to increase sensitivity. Briefly, fibrin-agarose indicator gels were prepared containing (final concentrations) 0.17 IU/mL bovine thrombin (Sigma), 25 µg/mL human plasminogen (Sigma), 0.05 IU/mL urokinase (Abbokinase, Abbott), 1.0% agarose, 2.3 mg/mL human fibrinogen (fraction I, essentially plasmin[ogen]-free; Sigma) in PBS. Immediately after they were mixed, the solutions were poured on the hydrophilic side of prewarmed agarose-gel support medium (Gelbond film, FMC Bioproducts) and allowed to solidify at room temperature. SDS-polyacrylamide gels were washed in 2x100 mL 2.5% Triton X-100 for 30 minutes each to remove SDS, rinsed in 2x100 mL distilled water for 1 minute each, placed on the fibrin-agarose gels, and incubated in a humid chamber at 37°C. After 3 to 4 hours, the fibrin-agarose gels had cleared (by the action of plasmin activated by urokinase), with the exception of opaque lysis-resistant zones. The gels were photographed in indirect light.
PAI-1 mRNA in Tissue
For extraction of total cellular RNA,
tissue samples were
pulverized under liquid nitrogen with a frozen stainless steel
crucible. The fine pulverized powder was transferred immediately to
RNAzol B (Tel-Test) and supplemented with chloroform (10% final
concentration). Subsequently, RNA was precipitated, washed, quantified,
size-fractionated, transferred to nylon membranes, and hybridized as
described previously.10 The integrity, equal loading, and
transfer of RNA in each lane were verified by ethidium bromide staining
of ribosomal RNA (rRNA).
A 0.9-kb fragment of human PAI-1 cDNA obtained
by digestion with
EcoRI and Sal I was labeled with deoxycytidine
5'-[-32P]triphosphate (Amersham), with random
oligonucleotides as primers (random primed DNA labeling kit, Boehringer
Mannheim). The labeled cDNA was used as a probe for hybridization with
PAI-1 mRNA. Human and rabbit PAI-1 cDNA hybridized similarly to rabbit
PAI-1 mRNA.
After hybridization, membranes were washed in 2xSSC (300 mmol/L sodium chloride, 30 mmol/L sodium citrate; pH 7.0) at room temperature for 5 minutes and subsequently in a solution containing 2xSSC and 1% SDS at 60°C for 90 seconds. These conditions were chosen to yield high-intensity PAI-1 signals and low background. Radioactivity in hybridized bands was delineated by autoradiography with Kodak XAR-5 film and intensifying screens (Cronex Lightning Plus, DuPont) at -70°C. Subsequently, radioactivity was quantified by laser densitometry (Ultroscan XL, Pharmacia LKB). Results in each case are averages in tissue of the same type from at least four rabbits.
Other Biochemical Procedures
Plasma concentrations of human
insulin were measured
conventionally by radioimmunoassay with second-antibody
precipitation.19 Plasma concentrations of human proinsulin
were measured with a novel radioimmunoassay that involves a
nonequilibrium binding reaction at room temperature plus polyethylene
glycol–assisted second-antibody precipitation.20
Fibrinogen in rabbit plasma was measured by the sulfite precipitation
method.21 TPA activity was assayed
spectrophotometrically.22
Statistical Analysis
Results are mean±SEM. The
significance of differences between
group means was assessed by ANOVA followed by Duncan's test (PAI-1
activity), one-way ANOVA (fibrinogen and TPA activity), and two-tailed
Student's t test for paired or unpaired observations as
appropriate. Significance was defined as P<.05.
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Results |
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Concentrations of Insulin and Proinsulin in Plasma
Insulin
In rabbits given vehicle alone, the concentration of insulin in plasma (detected with anti-human insulin antibody in the assay) remained virtually constant: 21.4±19.4 µU/mL at 0 hours, 9.2±5.2 µU/mL at the end of the infusion (1 hour), <8.0 µU/mL (limit of sensitivity) after 3 hours, and 9.6±5.8 µU/mL after 24 hours (n=3 each). In rabbits given human insulin over 1 hour, the corresponding values were 8.1±1.9, 688±142, 10.2±0.5, and 7.8±0.9 µU/mL (n=7 each). Thus, pharmacological concentrations of insulin in plasma were attained within 1 hour with a subsequent decline in 3 hours, which is consistent with observations by others.23 24
Proinsulin
In rabbits given human proinsulin over 1
hour, no proinsulin was
detectable, as expected, at 0 hours (limit of sensitivity, 0.001
nmol/L). After 1 hour of infusion, however, the concentration was
44.2±2.8 nmol/L (n=4 each), or comparable to that seen in
diabetic
patients treated with proinsulin.25 Although the infusion
mediums for insulin and proinsulin were equimolar (4.8±1.0 nmol/L),
concentrations of the two moieties in plasma after 1 hour differed
because of the fivefold more rapid clearance of insulin compared with
proinsulin (R.R. Bowsher, personal communication, 1993).
Activity of PAI-1 in Plasma
The time course of plasma
activity of PAI-1 was characterized in
23 rabbits over 24 hours; 7 rabbits were given insulin infused between
0 and 1 hour; 6, proinsulin; 4, C-peptide; and 6, vehicle alone.
Rabbits given insulin or proinsulin were maintained in a euglycemic
state by the clamp technique. In rabbits given vehicle alone or vehicle
plus C-peptide, blood glucose concentrations were monitored but no
infusion of glucose was needed or given.
PAI-1 activity changed
consistently with insulin and with proinsulin
(Fig 2). In control rabbits, PAI-1 activity increased
only slightly, probably as an acute-phase reactant in response to
procedures, but not significantly. In contrast, insulin elicited a
3.6-fold increase within 3 hours (P=.002 for all values from
2 to 6 hours). After 24 hours, PAI-1 was no longer significantly
elevated. Proinsulin increased PAI-1 significantly in 1 hour
(P=.001), with a peak 3.8-fold increase in 3 hours and a
return to baseline in 24 hours. Rabbits given C-peptide exhibited
changes indistinguishable from those in control rabbits. All values
were 7.4 AU/mL or lower.
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To determine whether the increase in plasma
PAI-1 activity was
attributable exclusively to an acute-phase reaction, fibrinogen was
assayed at 0, 3, and 24 hours (Table 1). After 3 hours,
concentrations of fibrinogen had not changed. After 24 hours, plasma
concentrations of fibrinogen had increased by approximately 60% in all
groups, as expected,22 probably in response to reduced
intake of fluid accompanying the use of tranquilizers and the
experimental procedures. However, no differences were seen between
groups. Another acute-phase reactant is TPA, with which activity
increases transiently, with a peak approximately 1 hour after infusion
of endotoxin in humans and rabbits.22 No early increase in
TPA activity was evident after administration of the agents used in
this study (Table 2). Thus, the effects of insulin and
proinsulin on PAI-1 activity (in contrast to the lack of an increase
with vehicle or C-peptide) do not appear to be simply manifestations of
an acute-phase reaction.
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Reverse Fibrin Autography
To define the nature of changes
underlying the inhibition of
plasminogen activator inhibiting activity, reverse fibrin autography
was performed. Fig 3 shows representative
results in rabbits given insulin, proinsulin, or vehicle alone.
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In
samples obtained 3 hours after the onset of infusions,
fibrinolysis-resistant zones were evident in regions indicative of
migration zones of a 75-kD protein, corresponding to
2-antiplasmin, which inhibits the action of plasmin and,
hence, lysis. Because the intensity in this zone was constant, changes
in 2-antiplasmin did not appear to account for the
increased inhibition of plasminogen activator activity seen with
insulin and proinsulin after 3 hours.
In plasma obtained 3 hours after the onset of infusions and subjected to immunoprecipitation of PAI-1, reverse fibrin autography showed lysis-resistant zones in a region corresponding to the migration zone of a 43-kD protein, consistent with immunoprecipitated and concentrated PAI-1. The molecular mass of rabbit PAI-1 is lower than that of human PAI-1 (47 to 50 kD, confirmed with purified human PAI-1 subjected to reverse fibrin autography under the same conditions). In plasma obtained 3 hours after administration of insulin and proinsulin, inhibition of fibrinolysis was much greater than that seen with samples taken at 0 hours, consistent with results of PAI-1 activity assays in plasma.
PAI-1 mRNA in Tissue
Additional rabbits were given insulin,
proinsulin, or vehicle
alone over 1 hour (n=4 each). In control rabbits, PAI-1 activity
increased by 1.4-fold (P=NS) within 3 hours, at which time
tissues were harvested. The increase was 4.1-fold with insulin
(P=.006) and 2.8-fold with proinsulin
(P=.004).
Rabbit tissues exhibited only one PAI-1 mRNA
species (3.2 kb),
consistent with previous observations.22 Rabbits given
insulin or proinsulin exhibited increased concentrations of PAI-1 mRNA
in aorta and liver 3 hours after the onset of infusions compared with
values in control rabbits. In aorta, the increase was 2.1-fold with
both insulin and proinsulin (n=4 each, P=.025). In
liver,
the corresponding increases were 2.4-fold (P=.001) and
1.9-fold (n=4, P=.015). No increases were seen in the
heart
(Fig 4). Preliminary results in additional rabbits
suggest that induction of PAI-1 gene expression occurred in para-aortic
adipose and skeletal muscle tissue as well with more pronounced
induction after proinsulin (data not shown).
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After 24 hours, the concentrations of PAI-1 mRNA in aorta and liver had returned to baseline (n=4 each). Thus, increased PAI-1 gene expression that may be accompanied by additional regulation on the translational level occurs in aorta and liver in 3 hours, corresponding to the time of maximal increase in plasma PAI-1 activity.
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Discussion |
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TopAbstractIntroductionMethods Results Discussion References |
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The results of the present study indicate that infusions of proinsulin or insulin significantly increase plasma PAI-1 activity but that infusions of C-peptide do not. These results are consistent with and extend our previous observations in vitro.10 With reverse fibrin autography, we found that the increases were attributable to PAI-1 protein. Increases induced by proinsulin and insulin were comparable, despite the fact that the concentrations of proinsulin in plasma were higher than those of insulin because of the more rapid clearance of insulin from the circulation. This is consistent with the lower affinity of proinsulin for the insulin receptor and the mediation of the proinsulin effect on PAI-1 synthesis through the insulin receptor, as shown previously in vitro.10 Increases in plasma PAI-1 activity were accompanied by increases in the concentrations of PAI-1 mRNA in aorta and liver. Thus, increased PAI-1 gene expression, with or without additional posttranscriptional regulation, appears to be responsible.
Rabbits were studied because of the known parallels between the fibrinolytic system and its regulation in rabbit and human systems.22 26 27 28 Proinsulin given intravenously to rabbits is not converted to insulin during the intervals involved in our study. Furthermore, <1% is converted to proinsulin split products.23 Endotoxin was excluded as a potential cause of the observed changes in plasma PAI-1 activity induced by proinsulin and insulin by monitoring all reagents with the Limulus assay. Because the time courses of changes in plasma fibrinogen of rabbits after insulin or proinsulin were similar to those in control rabbits, acute-phase reactions are unlikely to have accounted for the apparently specific effects on PAI-1 concentrations seen with proinsulin and insulin.
Despite the concordance of the present data with results in vitro, exogenous hyperinsulinemia under euglycemic and hyperglycemic conditions has not elicited increased plasma PAI-1 activity in human subjects in several studies.29 30 31 32 However, the subjects were studied in morning hours, when PAI-1 activity in plasma is known to be generally decreasing, in keeping with a typical diurnal pattern with peak values at 3 AM and a nadir at 6 PM.33 34 In addition, plasma concentrations of insulin induced in studies of normal human subjects29 30 31 were <100 µU/mL, a concentration exceeded in many patients with NIDDM and in rabbits in the present study. Conversely, in one of the studies, plasma insulin peaked with a concentration of 4400 µU/mL (28 nmol/L) in obese nondiabetic and obese diabetic patients.32 However, plasma PAI-1 was already high at baseline conditions in that study (51±8 ng/mL), possibly obscuring, attenuating, or precluding further increases in plasma concentrations of PAI-1.
PAI-1 activity in healthy volunteers and in obese patients given a high-calorie carbohydrate meal increased 2 hours later. Such transitory increases were preceded by a peak insulin response in 1 hour and appeared to modify the typical diurnal pattern with a decrease during the morning hours.35 These observations are consistent with the induction of peak concentration of insulin after 1 hour and a significant increase of plasma PAI-1 activity after 2 hours in the present study.
In patients with NIDDM, the activity of PAI-1 in plasma parallels the extent of fasting hyperinsulinemia, body mass index, and concentrations in plasma of triglycerides and apolipoprotein B. Most of these associations disappear after adjustment for insulin in multiple regression analyses.5 An association between plasma insulin and PAI-1 is evident also in patients with abdominal obesity36 and in those with systemic arterial hypertension.37 Interventions that reduce hyperinsulinemia, such as fasting for 24 hours or treatment with metformin (1,1-dimethylbiguanide) for 15 days,38 diminish plasma concentrations of PAI-1. Conversely, concentrations of proinsulin have been implicated in the increases of PAI-1 activity in plasma seen in patients with NIDDM.39
Increased plasma PAI-1 activity induced by proinsulin and insulin may contribute to a prothrombotic state. Increased endogenous PAI-1 promotes deposition of fibrin in ancrod-treated rabbits,28 exogenous PAI-1 prevents thrombolysis of jugular venous thrombi in rabbits,27 inhibition of PAI-1 activity by antibodies promotes endogenous and exogenous thrombolysis of such thrombi,26 and clot-bound PAI-1 suppresses endogenous fibrinolysis of pulmonary emboli in dogs.40 Increased plasma PAI-1 activity has been identified as a risk factor for deep venous thrombosis, coronary artery disease, recurrent acute myocardial infarction, and restenosis after angioplasty.10 41 42 Progression of coronary artery disease in patients with glucose intolerance43 is associated with elevated PAI-1 in plasma. Increased PAI-1 activity preceding pharmacological thrombolysis in patients with acute myocardial infarction presages a lower incidence of patency after treatment.44
Our results suggest that proinsulin and insulin, which are both found in increased concentrations of plasma in patients with NIDDM, abdominal obesity, and arterial hypertension, increase plasma PAI-1 activity and thereby potentially induce a prothrombotic state and accelerate atherosclerosis. Accordingly, attenuation of atherogenesis in subjects with hyperinsulinemia (hyperproinsulinemia) may be accomplished by normalizing concentrations of proinsulin and insulin in plasma, antagonizing effects of elevated concentrations of each on PAI-1 synthesis, or both.
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Acknowledgments |
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This article is dedicated to Prof Dr Wolfgang Kübler, Department of Cardiology, University of Heidelberg, Germany, on his 60th birthday. This study was supported in part by NIH grant HL-17646, Specialized Center of Research in Coronary and Vascular Diseases; Dr Thomas K. Nordt is supported by the Deutsche Forschungsgemeinschaft (No. 214/1-1). The authors thank Dr John A. Galloway, Lilly Research Laboratory, Eli Lilly Co, for human proinsulin; Dr Ronald Gingerich for measurements of plasma concentrations of insulin; Dr Ronald R. Bowsher of Lilly Research Laboratory for measurements of plasma concentrations of proinsulin; Pamela Lundius and John Engelbach for assistance with animal procedures; Denise Nachowiak, Jeffrey Labuda, and John Botz for biochemical assistance; and Lori Dales and Barbara Donnelly for secretarial support.
Received January 4, 1994; accepted September 23, 1994.
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