A Soluble PC-1 Circulates in Human Plasma: Relationship with Insulin Resistance and Associated Abnormalities1

Lucia Frittitta, Stefania Camastra, Roberto Baratta, Benedetta V. Costanzo, Monica D’Adamo, Salvatore Graci, Daniela Spampinato, Betty A. Maddux, Riccardo Vigneri, Eleuterio Ferrannini and Vincenzo Trischitta

Istituto di Medicina Interna, Malattie Endocrine e Metaboliche, Università di Catania, Ospedale Garibaldi (L.F., R.B., B.C., S.G., D.S., R.V., V.T.), 95123 Catania; Consiglionazionale della Ricerca, Institute of Clinical Physiology and Department of Internal Medicine, University of Pisa (S.C., E.F.), 56100 Pisa; Divisione ed Unita di Ricerca di Endocrinologia, Istituto Scientifico Ospedale Casa Sollievo della Sofferenza, San Giovanni Rotondo (V.T.), 71013 Foggia; and Divisione di Endocrinologia 1, Università La Sapienza (M.D.), 00100 Rome, Italy; and the Diabetes Research Laboratory, Mount Zion Hospital, University of California–San Francisco (M.D., B.A.M.), San Francisco, California 94143-1616

Address all correspondence and requests for reprints to: Lucia Frittitta, M.D., Endocrinologia, Ospedale Garibaldi, P.zza S. M. di Gesù, 95123 Catania, Italy. E-mail: segmeint{at}mbox.unict.it


    Abstract
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 Abstract
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 Experimental Subjects
 Materials and Methods
 Results
 Discussion
 References
 
An increased tissue content of PC-1, an inhibitor of insulin receptor signaling, may play a role in insulin resistance. Large scale prospective studies to test this hypothesis are difficult to carry out because of the need for tissue biopsies. The aim of this study was to investigate whether PC-1 is measurable in human plasma and whether its concentration is related to insulin sensitivity.

A soluble PC-1, with mol wt and enzymatic activity similar to those of tissue PC-1, was measurable in human plasma by a specific enzyme-linked immunosorbent assay and was inversely correlated to skeletal muscle PC-1 content (r = -0.5; P < 0.01). The plasma PC-1 concentration was decreased (P < 0.05) in insulin-resistant (22.7 ± 3.0 ng/mL; n = 25) compared to insulin-sensitive (36.7 ± 4.5; n = 25) nondiabetic subjects and was correlated negatively with the waist/hip ratio (r = -0.48; P < 0.001) and mean blood pressure (r = -0.3; P < 0.05) and positively with high density lipoprotein/total cholesterol (r = 0.38; P < 0.01) and both the M value and the plasma free fatty acid level decrement at clamp studies (r = 0.28; n = 50; P = 0.05 and r = 0.43; n = 22; P < 0.05, respectively). A plasma PC-1 concentration of 19 ng/mL or less identified a cluster of insulin resistance-related alterations with 75% accuracy.

In conclusion, PC-1 circulates in human plasma, and its concentration is related to insulin sensitivity. This may help to plan studies aimed at understanding the role of PC-1 in insulin resistance.


    Introduction
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 Abstract
 Introduction
 Experimental Subjects
 Materials and Methods
 Results
 Discussion
 References
 
INSULIN RESISTANCE is a characteristic feature of obesity and type 2 diabetes mellitus, but it is also present in up to 25% of healthy nonobese individuals (1, 2, 3, 4, 5, 6, 7, 8, 9, 10). A significant proportion of these individuals will eventually develop type 2 diabetes and/or coronary hearth disease (CHD) (1, 2).

The cellular mechanisms causing insulin resistance are not yet fully understood (3, 4, 5, 6, 7, 8, 9, 10). Recently, overexpression of several potential inhibitors of the insulin receptor tyrosine-kinase activity, a key step in insulin signaling, has been described in insulin-resistant subjects (11, 12, 13, 14). Although questioned by some researchers (15), these inhibitors include PC-1 (11), a class II (cytoplasmic amino-terminus) membrane glycoprotein with multiple enzymatic activities (pyrophosphatase and phosphodiesterase) that is expressed in many tissues and inhibits insulin signaling either at the level of the insulin receptor (11) or downstream at the level of the S-6 kinase activity (16). In both cell models and human tissues an elevated PC-1 content is associated with defective insulin receptor tyrosine kinase activity. In MCF-7 cells transfected with and overexpressing PC-1 there is a marked inhibition of insulin receptor tyrosine kinase activity and insulin action (11). In addition, PC-1 content is elevated in skeletal muscle, sc adipose tissue, and cultured skin fibroblasts of insulin-resistant nondiabetic subjects (17, 18, 19). In the same tissues, insulin receptor tyrosine kinase activity is impaired (17, 18, 19). These observations suggest that an elevated PC-1 content in insulin target tissues may play an important role in the development of insulin resistance. To test this hypothesis, large scale prospective studies are required. These studies, however, are difficult to perform because of the need for repeated tissue biopsies in a large number of subjects during a long observation period.

A soluble form of PC-1 has been detected in rat cell culture medium, rat serum (20), and human synovial fluid (21). Its biological significance is presently unknown. Whether a soluble form of PC-1 is present in human plasma and whether its concentration reflects insulin sensitivity are also unknown. If this is the case, however, soluble PC-1 measurement would considerably enhance the feasibility of large scale studies aimed at understanding the role of PC-1 in human insulin resistance and related abnormalities.

To address this issue, we set up a sensitive, specific enzyme-linked immunosorbent assay (ELISA) method to measure the PC-1 concentration in human plasma. We then studied 50 healthy subjects with a wide range of body mass indexes (BMIs) and insulin sensitivities.


    Experimental Subjects
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 Experimental Subjects
 Materials and Methods
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Fifty healthy subjects were studied after giving informed consent, according to the Declaration of Helsinki. None of them was diabetic [as determined by oral glucose tolerance test (OGTT)], and none was taking medications or had a clinically relevant disease. All subjects were consuming a weight-maintaining diet (50% carbohydrate, 30% lipid, and 20% protein) for the 3 weeks before the study. On the day of the study, after a 12-h fast, a blood sample was collected in the presence of NaK and ethylenediamine tetraacetate and centrifuged at 4 C. Plasma was immediately frozen at -20 C until PC-1 measurement. To quantitate plasma PC-1 fluctuations, three subjects were studied for 4 h (0900–1300 h) by collecting plasma samples every 30 min. Fasting plasma glucose was measured by the glucose oxidase method (Glucose Analyzer II, Beckman Coulter, Inc., Palo Alto, CA), and fasting plasma insulin was determined by an immunoenzymatic assay (IMx system insulin, Abbott Laboratories, Daimabot, Tokyo, Japan). Systolic and diastolic blood pressures were measured in all subjects using a standard mercury sphygmomanometer. Blood pressure was measured three times at 5-min intervals after the subject had rested supine for at least 10 min. All patients were normotensive (mean blood pressure, <107 mm Hg). The waist circumference was measured at the narrowest part of the torso, the hip circumference was measured in a horizontal plane at the level of the maximal extension of the buttocks, and the waist/hip circumference ratio was determined. The clinical and metabolic features of the subjects studied are shown in Table 1Go.


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Table 1. Clinical characteristics of the subjects studied

 

    Materials and Methods
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 Experimental Subjects
 Materials and Methods
 Results
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Euglycemic hyperinsulinemic clamp

Insulin-stimulated glucose disposal measurement by the euglycemic hyperinsulinemic clamp (22) was performed with a constant insulin infusion (40 mU/m2·min) and a variable glucose infusion to maintain plasma glucose within 10% of the baseline value. The M value (milligrams per kg BW/min) ranged from 2.2–11.1 (mean ± SE, 5.37 ± 0.21; median, 5.1). The 50 healthy subjects were subdivided into insulin-sensitive (M values >5.1, i.e. the median value) and insulin-resistant subjects (M value <5.1; Table 1Go).

Plasma PC-1 measurement

Wells in Maxisorb plates (Nunc, Roskilde, Denmark) were precoated (overnight incubation at 4 C) with an affinity-purified polyclonal antibody to PC-1 (provided by Dr. Yamashima). After washing with TBST buffer (20 mmol/L Tris, 150 mmol/L NaCl, and 0.05% Tween-20) to remove unbound antibody, wells were blocked with 150 mL TBST containing 1% BSA (30 min at 56 C) and washed again with TBST. Then, human plasma (10–30 µL diluted to a total volume of 100 µL with 50 mmol/L HEPES buffer, pH 7.6, containing 0.05% Tween-20, 1 mmol/L phenylmethylsulfonylfluoride, 2 mmol/L orthovanadate, 1% BSA, and 1 mg/mL bacitracin) was added to each well, and PC-1 was allowed to bind overnight at 4 C. After extensive washing with TBST, a biotinylated anti-PC-1 monoclonal antibody was added. After 2 h at 22 C, peroxidase-streptavidin was added, and after 30 min wells were washed again with TBST, and 100 µL biotinyl-tyramide solution were added. After 15-min incubation at 22 C, wells were washed with TBST, and streptavidin-horseradish peroxidase solution was added (30 min at 22 C). After further extensive washing, the peroxidase activity was determined colorimetrically by adding 3,3',5,5'-tetramethylbenzidine at a concentration of 0.4 g/L in an organic base and measuring the absorbance at 451 nm. In this assay, human plasma produced a dilution slope that paralleled the PC-1 standard purified as previously described (11) (Fig. 1Go). Intra- and interassay coefficients of variation were less than 10%.



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Figure 1. PC-1 ELISA. The PC-1 standard was purified from CHO cells transfected with and overexpressing human PC-1. Human plasma dilutions produced an inhibition-competition slope that paralleled that of the PC-1 standard.

 
Plasma PC-1 enzymatic activity

The phosphodiesterase activity of PC-1 was measured by the hydrolysis of thymidine 5'-monophosphate p-nitrophenyl ester (PNTP) (15). The reaction was carried out at saturating concentrations of the substrate by incubating 5–40 µL human serum with 1 µmol PNTP in 250 µL buffer (0.1 mol/L 2-amino-2-methyl-propanol and 7.5 mmol/L Mg(OAc)2, pH 9.4) for 60 min at 37 C. The reaction was stopped by the addition of 2 mL 0.1 N NaOH. Liberated p-nitrophenol was quantified spectrophotometrically by reading at 401 nm. Data were calculated as nanomoles of PNTP hydrolyzed in 1 h by 1 mL plasma.

Muscle PC-1 measurement

Muscle tissue specimens (external oblique) were obtained at elective abdominal surgery (cholecystectomy) from 27 additional healthy subjects. After adipose tissue was dissected, and blood was removed, specimens were immediately frozen in liquid nitrogen. Soluble extracts were subsequently prepared as previously described. Briefly, muscle tissue (~150 mg) was pulverized under liquid nitrogen and then homogenized in 2 mL buffer (50 mmol/L HEPES, 150 mmol/L NaCl, and 2 mmol/L PMSF, pH 7.6) at 4 C using a Polytron homogenizer (Brinkmann Instruments, Inc., Westbury, NY) for 10 s at medium speed. Triton X-100 was added to a final concentration of 1%, the homogenates were solubilized for 60 min at 4 C and centrifuged at 100,000 x g for 60 min at 4 C, and then supernatants were used for measurement of the PC-1 content (as described above), which was normalized for protein content (23).

Western blot studies

For Western blot analysis, human serum (~20–25 mL) was first applied to a wheat-germ agglutinin column, and the glycoproteins were eluted with acetylglucosamine 0.3 mol/L. Ten nanograms of PC-1 (measured by ELISA) were then immunoprecipitated from eluates with an affinity-purified anti-PC-1 polyclonal antibody coated with protein A-Sepharose for 16 h at 4 C. After centrifugation at 10,000 x g for 5 min, the pellet was washed three times with buffer [0.5 mol/L NaCl, 10 mmol/L sodium phosphate (pH 7.4), 0.5% Nonidet P-40, 2 mmol/L ethylenediamine tetraacetate, 0.1% SDS, and 0.04% BSA] and boiled in sample buffer [0.5 mol/L Tris-HCl (pH 6.8), glycerol, 10% SDS, 0.05% bromophenol blue, and ß-mercaptoethanol]. The sample was than centrifuged at 10,000 x g for 5 min, and the supernatant was subjected to SDS-PAGE in 7.5% polyacrylamide gel under reducing conditions. Proteins were transferred (2 h at 4 C) to nitrocellulose membranes that were first blocked with 10% BSA, washed with TBST buffer (10 mmol/L Tris, 150 mmol/L NaCl, and 0.5% Tween-20), and then incubated with 1 µg/mL anti-PC-1 monoclonal antibody. After 16 h at 4 C, membranes were incubated for 1 h with rabbit antimouse serum conjugated with horseradish peroxidase, and the reaction was developed with an enhanced chemiluminescence (ECL) detection system (Amersham International, Aylesbury, UK). The specific signaling was revealed by autoradiography.

Statistical analysis

Student’s t test and one-way ANOVA were used to compare mean values. Relationships between variables were evaluated by simple and multiple stepwise regression analysis. Data are presented as the mean ± SEM.


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Content and enzymatic activity of PC-1 in human plasma

When repeatedly measured every 30 min for 4 h in three subjects, the PC-1 plasma concentration did not change significantly, with observed variations being smaller than the intraassay coefficient of variation (data not shown). In the 50 subjects studied, the plasma PC-1 concentration ranged from 2.5–82.5 ng/mL (mean ± SEM, 29.7 ± 2.9 ng/mL; median, 26.0). No difference was observed between obese (BMI, >28 kg/m2) and nonobese (26.9 ± 4.4 and 31.0 ± 3.7 ng/mL, respectively) subjects.

Plasma PC-1 retained the pyrophosphatase activity typical of tissue PC-1 (17, 20). Pyrophosphatase activity was significantly (P < 0.01) higher in seven plasma specimens with high PC-1 (56 ± 6.1 ng/mL; range, 33.5–72) than in seven plasma specimens with low PC-1 (15 ± 3.1 ng/mL; range, 3.9–26.3): 28 ± 3 vs. 6 ± 1 nmol PNTP hydrolyzed/min·mL, respectively. When we measured plasma and muscle PC-1 in a subset of 27 healthy subjects, a negative correlation (r = -0.5; P < 0.01) between muscle (range, 4.5–82.5 ng/mg protein) and plasma (range, 2.5–20 ng/mL) PC-1 was observed (Fig. 2Go).



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Figure 2. Relationship between PC-1 content in plasma and in muscle specimens from 27 nonobese, healthy subjects (r = -0.5; P < 0.01).

 
PC-1 detection in human plasma by Western blot

When studied by PAGE and immunoblot, plasma PC-1 showed a similar mol wt as skeletal muscle PC-1 and PC-1 purified from Chinese hamster ovary (CHO) cells transfected with full-length human PC-1 complementary DNA (i.e. the standard PC-1 used in our ELISA; Fig. 3Go).



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Figure 3. PC-1 Western blot. Wheat-germ agglutinin eluates from human serum and solubilized extracts from two skeletal muscle specimens and CHO cells transfected with human PC-1 complementary DNA were immunoprecipitated with an anti-PC-1 polyclonal antibody, subjected to PAGE (7.5% polyacrylamide), transferred to a nitrocellulose filter, and blotted with an anti-PC-1 monoclonal antibody (see text for methods).

 
Relationship among plasma PC-1 concentration, insulin sensitivity, and related parameters

In the 25 insulin-sensitive healthy subjects, the mean PC-1 plasma level was significantly (P < 0.05) higher (36.7 ± 4.5 ng/mL) than that in the 25 insulin-resistant healthy subjects (22.7 ± 3.0 ng/mL; Fig. 4Go). Moreover, when all 50 subjects were considered together, the plasma PC-1 concentration was negatively correlated with both the waist/hip ratio (r = -0.48; P < 0.001; Fig. 5AGo) and mean blood pressure (r = -0.3; P < 0.05; Fig. 5BGo). In contrast, a positive correlation was observed with the high density lipoprotein (HDL)/total cholesterol ratio (r = 0.38; P < 0.01; Fig. 5CGo) and both the M value (r = 0.28; n = 50; P = 0.05) and the insulin-induced decrement in plasma FFA levels during the clamp (r = 0.43; n = 22; P = 0.05; Fig. 6Go, A and B). No significant correlation was observed between plasma PC-1 and BMI (r = 0.1; P = NS). When data were adjusted for BMI, sex, and age, plasma PC-1 was still correlated with both the waist/hip ratio and the decrement in plasma FFA (P < 0.05), but not with the other parameters.



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Figure 4. Plasma PC-1 concentration, as measured by ELISA, in healthy subjects either insulin sensitive (M at euglycemic clamp >5.1; n = 25) or resistant (M <5.1; n = 25). Data are the mean ± SEM. *, P < 0.05 vs. insulin-sensitive subjects.

 


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Figure 5. Relationships between plasma PC-1 concentration and waist/hip ratio (r = -0.48; P < 0.001; A), mean blood pressure (MBP; r = -0.3; P < 0.05; B), and HDL/total cholesterol ratio (r = -0.38; P < 0.01; C).

 


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Figure 6. Relationship between plasma PC-1 and 2 indexes of insulin sensitivity at euglycemic hyperinsulinemic glucose clamp: insulin-induced glucose disposal (M value; r = -0.28; P < 0.001; A) and insulin-induced decrement in plasma FFA levels ({Delta} FFA; r = -0.43; P < 0.05; B).

 
Finally, plasma PC-1 concentrations were not different at the beginning and the end of the glucose clamp in all subjects (27.4 ± 2.7 vs. 27.0 ± 2.7 ng/mL, before and after clamp, respectively) in either insulin-sensitive (33.1 ± 4.3 vs. 35.1 ± 4.0 ng/mL) or insulin-resistant (22.7 ± 3.2 vs. 19.8 ± 3.0 ng/mL) subjects analyzed separately.

Plasma PC1 levels and the clustering of the insulin resistance syndrome

Looking for the cluster of insulin resistance syndrome, tertiles were obtained for the following parameters (plasma glucose at 0 and/or 120 min of OGTT, plasma insulin at 0 and/or 120 min of OGTT, serum triglycerides, HDL/total cholesterol ratio, and mean blood pressure). Clustering was defined as four or more of the mentioned parameters being in the worst tertile (the highest for glycemia, insulinemia, triglyceridemia, and mean blood pressure and the lowest for HDL/total cholesterol ratio). When the 50 healthy subjects were subdivided into tertiles according to their plasma PC-1 concentration (range, 2.5–17, 18.5–34, and 35–82.5 ng/mL in tertiles 1, 2, and 3, respectively), the cluster of insulin resistance was more frequent in subjects from tertile 1 (7 of 17, 41%) than in tertiles 2 (1 of 16, 6.3%) and 3 (1 of 17, 5.9%; P < 0.05, by ANOVA). A plasma PC-1 level of 19 ng/mL or less identified this cluster of alterations typical of insulin resistance with 75% accuracy (75% sensitivity and 75% specificity).


    Discussion
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 Experimental Subjects
 Materials and Methods
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We have identified a soluble PC-1 form that circulates in human plasma and retains enzymatic phosphodiesterase activity. In 50 healthy subjects with a wide range of insulin sensitivity and BMI, a low plasma PC-1 concentration was significantly associated with phenotypic expression of aggregated features of the insulin resistance syndrome, including relatively low insulin sensitivity, high waist/hip ratio, high blood pressure, and low HDL/cholesterol ratio. Subjects with a low plasma PC-1 concentration, therefore, could be at a higher risk of developing type 2 diabetes and/or CHD. An important additional issue is whether low plasma PC-1 could be a predictor for the future development of insulin resistance and related abnormalities in healthy individuals who, at present, do not show the cluster of insulin resistance-related abnormalities. A longitudinal study, which has been, in fact, undertaken in our laboratory is needed to answer this question.

Previous studies have shown that in insulin-resistant subjects, the PC-1 content is increased in insulin target tissues (including muscle and adipose tissues) (17, 18, 19, 24) and that the PC-1 may act through inhibition of the insulin receptor tyrosine kinase activity. The reason why insulin-resistant individuals have an increased PC-1 content in tissues but a reduced PC-1 concentration in plasma is not known. One possibility is that the PC-1 content is increased in tissues because its release into extracellular fluids is impaired, as suggested by the negative correlation we observed between plasma and skeletal muscle PC-1 content. Another possibility is that in insulin-resistant subjects, circulating PC-1 is cleared at a higher rate from plasma (either bound or degraded).

The observation that the PC-1 plasma concentration is not affected by the acute hyperinsulinemia obtained in the glucose clamp study is in concert with the recent observation that the PC-1 tissue content is not affected by chronic hyperinsulinemia in patients with insulinoma (25) and suggests that hyperinsulinemia does not regulate PC-1 tissue content and/or release.

PC-1 released from rat cultured cells has a smaller mol wt than cellular PC-1, and it is likely to be cleaved intracellularly, with no evidence of plasma membrane-associated cleavage (20). In contrast, human plasma PC-1 has a mol wt similar to that of tissue PC-1. This finding is compatible with either the active secretion of intact PC-1 from human tissues or the binding into plasma of cleaved PC-1 to an unknown circulating factor(s).

Cell membrane PC-1 as well as other ecto-enzymes may hydrolyze nucleotides to nucleosides, which subsequently are taken up by cells through nucleoside transporters (26). This would act as a salvage pathway for those cells that have a low capacity to synthesize purine nucleotides by collecting the presynthesized nucleosides from the extracellular fluid. As human plasma PC-1 retains its phosphodiesterase activity, it may contribute to the metabolism of purine nucleotides.

Whatever its biological significance, plasma PC-1 is significantly related to several features of the insulin resistance syndrome. If a cause-effect relationship between PC-1 and the insulin resistance syndrome is established, measuring plasma PC-1 may easily identify subjects with PC-1-related insulin resistance. Breaking up such a heterogeneous condition into distinct phenotypes may be the key for future treatment of insulin resistance and its clinical outcomes (i.e. type 2 diabetes and/or CHD).


    Footnotes
 
1 This work was supported by a grant from Ministero dell’ Università e della Ricerca Scientifica e Tecnologica (MURST 60%; to R.V.). Back

Received May 6, 1999.

Revised June 22, 1999.

Accepted June 29, 1999.


    References
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 Abstract
 Introduction
 Experimental Subjects
 Materials and Methods
 Results
 Discussion
 References
 

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