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Published online June 11, 2007
Diabetes 56:2028-2035, 2007
DOI: 10.2337/db07-0394
© 2007 by the American Diabetes Association
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Soluble Insulin Receptor Ectodomain Is Elevated in the Plasma of Patients With Diabetes

The Soluble Insulin Receptor Study Group*

Address correspondence and reprint requests to Yousuke Ebina, MD, PhD, Division of Molecular Genetics, Institute for Enzyme Research, The University of Tokushima, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan. E-mail: ebina{at}ier.tokushima-u.ac.jp

Abbreviations: ELISA, enzyme-linked immunosorbent assay; FPG, fasting plasma glucose; hIR, human insulin receptor; IL, interleukin; IR, insulin receptor; IRI, immunoreactive insulin; MMP, membrane-tethered zinc metalloproteinases; PKC, protein kinase C; STZ, streptozotocin; TNF, tumor necrosis factor; WGA, wheat-germ agglutinin


   ABSTRACT
TOP
 ABSTRACT
RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX
 REFERENCES
 
OBJECTIVE—Insulin binds to the {alpha}-subunit of the insulin receptor (IR{alpha}) and subsequently exerts its effects in the cells. The soluble ectodomains of several receptors have been found to circulate in the plasma. Therefore, we hypothesized that soluble human insulin receptor (hIR) ectodomain ({alpha}-subunit and a part of ß-subunit) may exist in the plasma of diabetic patients.

RESEARCH DESIGN AND METHODS—We identified soluble hIR ectodomain in human plasma by a two-step purification followed by immunoblotting and gel-filtration chromatography. Furthermore, we established an hIR{alpha}-specific enzyme-linked immunosorbent assay and measured the plasma IR{alpha} levels in patients with diabetes. We also investigated this phenomenon in streptozotocin-induced diabetic hIR transgenic mice.

RESULTS—Soluble hIR{alpha}, but not intact hIRß or whole hIR, exists in human plasma. The plasma IR{alpha} levels were significantly higher in type 1 (2.00 ± 0.60 ng/ml; n = 53) and type 2 (2.26 ± 0.80; n = 473) diabetic patients than in control subjects (1.59 ± 0.40 ng/ml; n = 123 (P < 0.001 vs. control). Plasma IR{alpha} level was positively correlated with blood glucose level, and 10–20% of the insulin in plasma bound to hIR{alpha}. In the in vivo experiments using diabetic hIR transgenic mice, hyperglycemia was confirmed to increase the plasma hIR{alpha} level and the half-life estimated to be ~6 h.

CONCLUSIONS—We propose that the increased soluble IR ectodomain level appears to be a more rapid glycemic marker than A1C or glycoalbumin.

The ectodomains of receptors for several cytokines and growth factors have been found to circulate in plasma (1–3). In 1972, Gavin et al. (4) demonstrated that an insulin binding protein was shed from the surface of cultured cells. Subsequently, Pezzino et al. (5) observed a circulating protein that corresponded to the insulin receptor (IR) in healthy human plasma. Furthermore, the IR {alpha}-subunit (IR{alpha}) and IR ß-subunit (IRß) were found to be secreted into the incubation medium by various cultured cell lines (6), and IR shedding from cultured human lymphocytes has been reported (7). Thus, the existence of soluble IR in human serum has been suspected. However, no detailed clinical investigation has yet been carried out. We previously reported that an injection of purified human insulin receptor (hIR) {alpha}-subunit (hIR{alpha}) increased the blood glucose level in mice (8). Furthermore, transgenic mice secreting soluble IR{alpha} into the plasma showed chronic hyperglycemia (9). Here, we established novel enzyme-linked immunosorbent assay (ELISA) systems to measure both the ectodomain ({alpha}-subunit and a part of ß-subunit) of IR and full length of IR. With these ELISA systems, we report that soluble hIR{alpha} with parts of extracellular region of hIRß, but not as a whole IR or with intact hIRß, is present in human plasma and that its plasma level is elevated in patients with elevated blood glucose. The ectodomain of IR may be cleaved, at least in part, by hyperglycemic state–associated mechanisms.


   RESEARCH DESIGN AND METHODS
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
RESULTS
 DISCUSSION
 APPENDIX
 REFERENCES
 
Insulin receptor sandwich ELISA systems.
We developed two kinds of ELISA systems to specifically measure hIR{alpha} and full-length IR, respectively (details available in an online appendix at http://dx.doi.org/10.2337/db07-0394).

Study subjects.
Healthy Japanese volunteers with no diabetic history or familial diabetic history (in relatives within the third degree) residing in the Tokushima district (n = 123) and confirmed to have normal glucose tolerance by 75-g oral glucose tolerance according to the World Health Organization guidelines for diabetes (10) were enrolled as control-1 subjects. Healthy Japanese volunteers with no diabetic history or familial diabetic history residing in the Nagano district (n = 120) and confirmed to have normal fasting plasma glucose (FPG) and A1C levels were enrolled as control-2 subjects. Outpatients seen at the Shiga University of Medical Science in the Shiga district (type 2 diabetic, n = 474), the University of Tokushima Affiliated Hospital in the Tokushima district (type 2 diabetic, n = 162), and the Tokushima University Hospital in the Tokushima district (type 1 diabetic, n = 53) were enrolled as T2DM-1, T2DM-2, and T1DM patients, respectively.

Laboratory measurements.
All clinical laboratory data for the T2DM-1 and T1DM patients were obtained at the Central Clinical Laboratory of the University Hospital of Shiga University of Medical Science or the Central Clinical Laboratory of the University of Tokushima. Plasma glucose was measured by the glucose oxidase method. Insulin and urine C-peptide immunoreactivity were estimated using ELISA methods, while the A1C level was measured by high-performance liquid chromatography. Total cholesterol, triglycerides, HDL cholesterol, free fatty acid, lactate, and glycoalbumin levels were determined using standard enzymatic methods.

hIR-expressing transgenic mice.
In this study, we used two strains of transgenic mice expressing hIR (details in online appendix).

Immunoblotting.
Immunoblotting was carried out using specific antibodies as indicated in the figure legends and as previously described except using Can-get-signal solution (Toyobo, Osaka, Japan) for primary antibody dilution (8).

Reagents.
All other reagents were of analytical grade and obtained from Sigma or Nacalai Tesuque (Kyoto, Japan).

Ethical issues.
All study protocols and designs were approved by the ethics committees of the University of Tokushima (approval #171) and/or the Shiga University of Medical Science (approval #16–36). We also obtained written informed consent from all participants. Animal experiments were approved by the animal ethics committees of the University of Tokushima (approval #16–57 and #05052) and carried out in accordance with international care regulations.


   RESULTS
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
DISCUSSION
 APPENDIX
 REFERENCES
 
Detection of soluble IR ectodomain in human plasma.
Based on the hypothesis that soluble IR{alpha} may exist in human plasma, we attempted to detect IR{alpha} in human plasma. After partial purification by wheat-germ agglutinin (WGA)-conjugated affinity chromatography (GE Healthcare Bio-Sciences, Piscataway, NJ) and immunoprecipitation with an anti–hIR{alpha}-specific antibody (5D9), IR{alpha} (135 kDa) was detected by immunoblotting with an IR{alpha}-specific antibody (N-20). A band corresponding to hIR{alpha}, but not the IR precursor, was observed, and its peak signal intensity corresponded to the peak absorbance in hIR{alpha} ELISA analyses (A450) (Fig. 1A). Furthermore, intact hIRß was not detected by immunoblotting with an anti-IRß antibody that recognized the COOH-terminal 19 amino acids of IRß (data not shown), suggesting that the hIR{alpha} observed in human plasma was probably derived by the cleavage of the ectodomain ({alpha}-subunit and a part of ß-subunit) of the receptor from cell surfaces, rather than by release from damaged tissues (cells) or microvesicles (online appendix Fig. 3 and in-text Table 1). Furthermore, we also compared the molecular weights of the soluble hIR ectodomain in human plasma with standard hIR-ectodomain protein ({alpha}-subunit and a part of ß-subunit) (8). The standard protein was derived from CHO-IR-SspI cells (8) and stably introduced the cDNA encoding the whole hIR ectodomain except for just three amino acids of the peritransmembrane region (amino acids 1–953), and its logical molecular weight was ~370 kDa (8). As shown in Fig. 1B, the retention time of immunoreactive hIR ectodomain in human plasma was identical to that of standard hIR-ectodomain protein, suggesting that the molecular weight of both proteins under nonreducing conditions were almost same using the Superdex-200 gel-filtration column. In addition, the apparent molecular weight was ~370 kDa. If the soluble hIR ectodomain exists as {alpha}-subunit homodimers, the molecular weight would be ~270 kDa. If the soluble hIR{alpha} exists with intact ß-subunits as an intact heterotetramer, the molecular weight would be ~460 kDa. Given all these data, the soluble hIR{alpha} appeared to exist with parts of the extracellular region of ß-subunits, and proteolytic cleavage ("shedding") appeared to occur at a site in the extracellular peritransmembrane region (online appendix Fig. 3).


Figure 1
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  		FIG. 1. A: Detection of soluble IR{alpha} in human plasma. A 10-ml sample of human plasma (hIR{alpha} ~5 ng/ml) obtained from a patient was diluted to 50 ml with phosphate salt (PS) buffer (20 mmol/l phosphate buffer, pH 7.4, containing 0.5 mol/l NaCl) and applied to a WGA column (2-ml bed volume). The column was washed with PS buffer and eluted with the same buffer supplemented with 0.3 mol/l N-acetyl-glucosamine. Aliquots (300 µl) of the 1-ml fractions obtained were subjected to immunoprecipitation (IP) with the 5D9 antibody and analyzed by immunoblotting (IB, reducing condition) with an anti-IR{alpha} antibody (N-20). The titer of hIR{alpha} in each fraction obtained from the WGA column chromatography was assayed by the hIR{alpha} ELISA and is shown as the absorbance at 450 nm (A450). B: Comparison of the native molecular weights of human plasma soluble IR and standard IR ectodomain protein using Superdex 200 column. A 0.5-ml sample of human plasma obtained from a normal subject and standard hIR ectodomain protein for the ELISA system were diluted to 0.5 ml with column buffer (10 mmol/l phosphate buffer, pH 7.0, containing 0.1 mol/l NaCl, 0.1% BSA, and 0.1% NaN3) and applied to a Superdex 200 gel-filtration column (1.5 x 60 cm; GE Healthcare Bio-Sciences). The column was eluted with same column buffer. Aliquots (100 µl) of the 1.0-ml fractions obtained were subjected to hIR{alpha}-specific Sandwich ELISA. The titer of hIR{alpha} (ng/ml) and the absorbance at 280 nm (A280) in each fraction obtained from either human plasma (•) or standard hIR ectodomain protein ({blacktriangleup}) are shown. The approximate molecular weight of IgG (150 kDa) and albumin (67 kDa) in the human plasma sample are also shown. The eluted fractions showed the analytical recovery of immunoreactive plasma soluble hIR and standard hIR ectodomain to be 87 and 97%, respectively.

 

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  		TABLE 1 Comparisons of the plasma hIR{alpha}, intact IRß, and full-length hIR levels in patients with diabetes

 
Measurements of soluble IR{alpha} in human plasma from diabetic patients using the newly established hIR{alpha}-specific ELISA system.
Based on the above results, we established ELISAs for both hIR{alpha} and full-length hIR that were highly accurate, specific, and unaffected by the presence of either insulin or hemolysis (details in RESEARCH DESIGN AND METHODS). According to the receiver operator characteristic analysis using the data of control-1 and T2DM-1 groups, the area under the curve was 0.794 ± 0.217 (P < 0.0001, online appendix Fig. 1G), suggesting that this hIR{alpha} ELISA system has significant specificity and sensitivity in separating these two groups. We also used a commercially available IRß ELISA system, as well as a full-length IR ELISA, to confirm that the plasma hIR{alpha} was not derived from damaged cells or microvesicles. To investigate the clinical significance of the presence of IR{alpha} in human plasma, we measured the hIR{alpha} levels in samples from patients with diabetes. First, we examined the variations in the hIR{alpha} level in normoglycemic individuals throughout the day and found that the level did not change in normoglycemic subjects, even in the postprandial state (online appendix Fig. 1F). Next, we measured plasma samples obtained from both the T2DM-1 and T2DM-2 groups, as well as from the control-1 group, who had been confirmed to be normoglycemic by oral glucose tolerance tests according to World Health Organization criteria (10). As shown in Fig. 2A, the T2DM-1 group exhibited a significantly elevated plasma hIR{alpha} level compared with the control-1 group (2.26 ± 0.80 [n = 474] vs. 1.59 ± 0.40 ng/ml [n = 123], respectively; P < 0.001). On the other hand, the levels of plasma intact IRß and full-length IR were negligible in the type 2 diabetes plasma samples with a high hIR{alpha} level (Table 1), indicating that the plasma hIR{alpha} existed without intact IRß and was derived from the cell surface by cleavage rather than by release from damaged cells. These results are also supported by Fig. 1B. The plasma hIR{alpha} level was also increased in the T1DM group (2.00 ± 0.60 ng/ml [n = 53], P < 0.001) versus the control-1 group (Fig. 2A), and the levels of plasma intact IRß and full-length IR were also negligible (Table 1). More than 30% of the diabetic patients exhibited an hIR{alpha} level higher than the cutoff value (2.39 ng/ml, mean + 2 SD of control-1 group values). There were no differences in the hIR{alpha} levels among people from different districts. On a molar basis, the hIR{alpha} concentration in diabetic patients was ~20% of the fasting plasma insulin concentration. We also evaluated the percentages of insulin-bound hIR{alpha} in plasma from 11 cases of type 2 diabetic patients by measuring the changes in the immunoreactive insulin (IRI) level after depletion of hIR{alpha} by 5D9 (anti-hIR{alpha})-affinity beads. Under conditions of almost complete hIR{alpha} depletion, a comparable amount (~10–20%) of the IRI was depleted by the anti-hIR{alpha} antibody beads. Therefore, at most, 10–20% of the IRI binds to the plasma hIR{alpha} (Table 2).


Figure 2
Figure 2
Figure 2
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  		FIG. 2. Plasma hIR{alpha} levels in patients with type 1 and type 2 diabetes. A: Comparisons of the plasma IR{alpha} levels among patients with diabetes and normoglycemic control subjects. Data are expressed as the mean ± SD. The cutoff value (2.39 ng/ml) was determined by the mean + 2 SD of the control-1 value. ***P < 0.001 by Student's t test. B: Scatter plot showing the correlation between changes in plasma hIR{alpha} level and changes in A1C level over 36 months (March 2000–March 2003) in selected ({Delta}A1C ≥1.8) T2DM-1 patients (n = 18). The correlation coefficient was determined by Pearson's correlation coefficient test. C: Clinical courses of eight new-onset type 1 diabetic patients. The hIR{alpha}, FPG, glycoalbumin (GA), and A1C levels (mean ± SD) at admission, discharge (6–10 days after admission) and 1- and 2-month follow-up are shown. D: Scatter plot showing correlation of plasma hIR{alpha} level with A1C, glycoalbumin, and plasma glucose in 118 samples from 64 type 1 diabetic patients. The correlation coefficients were determined by Peason's correlation coefficient test for glycoalbumin, plasma glucose, and A1C. E: The serial changes of glycemic control markers in a type 1 diabetic patient. The glycemic markers (A1C {blacksquare}, glycoalbumin {square}, and hIR{alpha} {triangleup}) of a type 1 diabetic outpatient seen at the University of Tokushima Hospital are plotted.

 

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  		TABLE 2 Insulin binding by hIR{alpha} in human plasma from 11 type 2 diabetic patients

 
Next, we analyzed the correlations of the hIR{alpha} level with the clinical parameters of the diabetic patients (online appendix Table 1). There were significant positive correlations between the hIR{alpha} and the blood glucose levels, including fasting blood glucose (P < 0.001) and A1C (P < 0.001) levels (online appendix Table 1 and online appendix Fig. 2A and B). On the other hand, the hIR{alpha} level was not correlated with markers of insulin secretion, including fasting IRI level, 24-h urinary C-peptide immunoreactivity excretion, or homeostasis model assessment of ß-cell function. The hIR{alpha} level also exhibited a very weak correlation with age (P < 0.001) in the T2DM-1 group, but not in the control-1, control-2, or T2DM-2 groups. As shown in online appendix Table 1, there were no correlations with other standard clinical parameters. Moreover, there were no significant differences in the hIR{alpha} levels among the types of treatments the patients were receiving. To further confirm the relationship between hIR{alpha} and blood glucose levels, we examined 18 patients whose A1C level had changed greatly (≥1.8%) within the last 3 years. As shown in Fig. 2B, there was a strong positive correlation between the changes in the plasma hIR{alpha} level and those in the A1C level (n = 18, P < 0.001, R = 0.84). To examine whether hyperglycemia induced the increase in plasma hIR{alpha}, we followed the clinical courses of eight new-onset type 1 diabetic patients (Fig. 2C), all of whom were sent to the Tokushima University Hospital due to the onset of type 1 diabetes (T1DM group). At admission, FPG, A1C, and hIR{alpha} levels were 350 ± 82 mg/dl, 12.6 ± 2.1%, and 5.5 ± 1.8 ng/ml, respectively. At discharge after 6–10 days of intensive insulin therapy, their blood glucose levels had almost normalized (124 ± 31 mg/dl) and hIR{alpha} levels had decreased to 3.3 ± 1.0 ng/ml. After 1 month, the patients' glycemic control levels were maintained with insulin therapy and their A1C, glycoalbumin, and hIR{alpha} levels were 9.2 ± 1.3%, 24.9 ± 2.4%, and 2.3 ± 0.8 ng/ml, respectively. At that moment, only hIR{alpha} almost reached plateau, but glycoalbumin and A1C levels were further decreased at 2 months. The results presented in Figs. 2B and C strongly suggest that plasma hIR{alpha} levels promptly parallel any changes in blood glucose levels and changed more rapidly than glycoalbumin or A1C. In addition, there was a significant positive correlation of hIR{alpha} levels with blood glucose levels including blood glucose, glycoalbumin, and A1 C (online appendix Table 1, in-text Fig. 2D, online appendix Fig. 2A, and online appendix Fig. 2B). The regression values were higher in the type 1 diabetic patients (Fig. 2D) than in the type 2 diabetic patients (online appendix Figs. 2A and B). Furthermore, during long-term follow-up of a type 1 diabetic patient, hIR{alpha} level completely paralleled with glycoalbumin and A1C levels and changed more rapidly and dynamically than glycoalbumin and A1C levels (Fig. 2E).

Elevation of the plasma hIR{alpha} level in streptozotocin-induced diabetic mice transgenically expressing hIR.
To further confirm these clinical observations in vivo, we initially used transgenic (TG) mice that systemically express kinase-deficient hIR (hIRK1030MTG) (11,12). Although these TG mice express a kinase-deficient mutant hIR (K1030M; Lys->Met at residue 1,030 in the kinase domain of the ß-subunit), they do not show diabetic phenotypes (11,12). Furthermore, the mutation did not appear to affect the receptor release based on analyses of cultured cells (data not shown). In addition, we recently generated and analyzed wild-type (WT) hIR–expressing TG mice that systemically expressed at least fourfold higher amounts of hIR than hIRK1030MTG mice (data not shown).

Diabetes was induced in either TG or control mice (nontransgenic [NTG] littermates) by intraperitoneal streptozotocin (STZ) injection. After 4–7 days, diabetic hIR-TG mice showed a markedly higher hIR{alpha} level, and this was strongly correlated with their blood glucose levels (n = 14, P < 0.001, R = 0.80; Fig. 3C). On the other hand, NTG diabetic mice or nondiabetic hIR-TG mice showed negligible hIR{alpha} levels, similar to those of NTG nondiabetic mice. In the NTG diabetic mice, the endogenous mouse IR{alpha} level was expected to increase but was not detected with the hIR{alpha}-specific ELISA systems. The level of full-length IR was also confirmed to be negligible using these ELISA systems, indicating that the plasma hIR{alpha} was not derived from damaged cells (Fig. 3B). Furthermore, to investigate whether the IR{alpha} responses were attributed to the changes of glucose level, we treated the STZ-induced diabetic mice with insulin (subcutaneous injection twice a day of NPH human insulin, Novolin-N; NovoNordisk, Bagsv\jrd, Denmark). As shown in Fig. 3D, the IR{alpha} levels were promptly decreased by insulin therapy paralleled with blood glucose level. Moreover, after a transient pause (3 days) of insulin treatments, both blood glucose and IR{alpha} levels were reelevated and then promptly redeclined by the resumption of insulin therapy. Notably, some mice failed to develop diabetes even with same dose of STZ and did not exhibit elevated IR{alpha} levels. We also treated nondiabetic mice with sustained-release insulin implants (Linshin Canada, Toronto, ON, Canada), and these mice showed normoglycemia and marked weight gain, but the IR{alpha} levels were changed little (data not shown), indicating that neither STZ nor insulin alone changed plasma IR{alpha} levels. We further estimated the half-life of the IR{alpha} in blood using the WT-hIR-TG mice. After diabetes induction by STZ, insulin therapy was initiated and plasma IR{alpha} levels decreased, parallel with glucose levels. As shown in Fig. 3E, the circulating plasma IR{alpha} levels decreased to 49.3 ± 13.8% (n = 9) at 6 h after the treatment, 28.7 ± 17.5% (n = 19) at 24 h, and 19.0 ± 12.3% (n = 19) at 48 h.


Figure 3
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  		FIG. 3. Detection of hIR{alpha} in STZ-induced diabetic mice expressing hIR. A and B: STZ (4 mg/20 g body wt) was injected intraperitoneally into 6-week-old fasted TG mice systemically expressing kinase-deficient hIR (hIRK1030MTG) (29) or control NTG littermates (C57BL/6 strain). After 4–7 days, blood samples were obtained and analyzed by the hIR{alpha} (A) and full-length IR (B) ELISA systems. •, STZ-injected mice; {circ}, vehicle (50 mmol/l citrate)-injected mice. Mice with a glucose level of ≥300 mg/dl were assigned to a diabetic group. ***P < 0.001 vs. the other groups by Bonferroni-Duncan's multiple comparison test. C: Scatter plot showing the correlation between the plasma hIR{alpha} level and the blood glucose level in STZ-induced hIR-TG mice (hIRK1030MTG). The correlation coefficients were determined by Pearson's correlation coefficient test. D: Serial changes of STZ-induced hIR-WT-TG mice with insulin treatment. Three days after the induction of diabetes with STZ, the mice were treated with NPH insulin (6–14 units/day, twice a day subcutaneous injection), then with a 3-day transient pause of insulin treatments, followed by resuming insulin therapy. Blood glucose levels were monitored twice a day, and insulin doses were determined by glucose levels. E: The estimation of half-life of hIR{alpha} and ß in plasma. Three days after the induction of diabetes with STZ, the diabetic hIR-WT-TG mice were treated with NPH insulin (6–14 units/day, twice a day subcutaneous injection). Circulating plasma hIR{alpha} and ß levels were monitored at 0, 6, 24, and 48 h after initiation of insulin therapy.

 

   DISCUSSION
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
APPENDIX
 REFERENCES
 
In this study, we provide evidence that soluble hIR ectodomain ({alpha}-subunit and a part of ß-subunit), but not intact hIRß or whole hIR, exists in human plasma. Furthermore, patients with type 2 diabetes, as well as those with type 1 diabetes, showed a significantly elevated plasma hIR{alpha} level compared with control subjects, as measured using newly established hIR-specific ELISA systems (i.e., hIR{alpha}-specific and whole hIR–detectable systems). Plasma hIR{alpha} level positively correlated with blood glucose, glycoalbumin, and A1C levels. In addition, comparable amounts of plasma insulin appeared to bind to hIR{alpha}. Moreover, hyperglycemia was confirmed to induce hIR{alpha} release in STZ-induced diabetic mice transgenically expressing hIR.

The ectodomains of receptors for several cytokines and growth factors have been found to circulate in plasma (1–3). The existence of soluble IR in human plasma has previously been suspected, since several studies have reported shedding of IR from cultured cells (e.g., IM-9 human lymphoblasts, MCF-7 human breast cancer cells, HepG2 human hepatoma cells, and human lymphocytes, as well as 3T3-L1 mouse fibroblasts transfected with hIR) (4,6,7). We have also observed hIR{alpha} release from CHO-hIR cells, HepG2 cells, hIR-expressing L6 myocytes, and hIR-expressing 3T3-L1 adipocytes (T. Obata, K. Yokoyama, E. Okamoto, T. Yuasa, Y. Ebina, unpublished data).

Although Pezzino et al. (5) reported the detection of both IRß and IR{alpha} in healthy human plasma that presented insulin-stimulated autophosphorylation activity without tyrosine kinase activity against exogenous substrates, we did not observe a protein corresponding to the ß-subunit, at least in its intact form, in either of the ELISA (full-length and ß-subunit) systems or in immunoblotting (data not shown). We showed that IR{alpha} is released concomitantly with part of the extracellular domain of IRß (online appendix Fig. 3) into the plasma of diabetic patients using Superdex gel-filtration column (Fig. 1B). In fact, many membrane proteins (e.g., tumor necrosis factor [TNF] receptor, epidermal growth factor receptor, and interleukin [IL]-6 receptor) have soluble ectodomains that are usually cleaved at a site in the stalk region between the transmembrane segment and the globular extracellular domain. The distances of the cleavage sites from the plasma membrane are ~1–43 amino acids (13). In accordance with this report, as shown in Fig. 1B, the retention times of immunoreactive soluble hIR in human plasma and standard hIR ectodomain protein derived from CHO-IR-SspI cells were similar, suggesting that the molecular weight of both proteins under the nonreducing condition were almost same. Moreover, the apparent molecular weight was ~370 kDa. Considering all of this information, the ectodomain shedding appeared to occur at a site in the stalk region of the extracellular peritransmembrane region, as in the case of other soluble receptors; as a result, the soluble IR{alpha} in human plasma appeared to exist with parts of the extracellular region of ß-subunits (online appendix Fig. 3). Since the actual cleavage site is unclear at this time, further experiments are necessary. Recently, the crystal structure of IR-ectodomain has been clarified (14), therefore these data must be helpful.

In the present study, the clinical results indicated that the release of hIR{alpha} into plasma may be augmented by hyperglycemia, and the subsequent in vivo study using hIR-TG mice supports these findings. On the other hand, plasma hIR{alpha} level was not correlated with markers of insulin secretion, suggesting that the release of hIR{alpha} into plasma may be regulated by blood glucose level, rather than secreted insulin. The concentration of hIR{alpha} in patients with diabetes was on average ~20% (20.3 ± 51.2%, n = 88) of the fasting IRI levels on a molar basis. Comparable amounts (~10–20%) of insulin were immunodepleted by anti-IR{alpha} antibody (Table 2), suggesting that appreciable amounts of plasma insulin appeared to bind to plasma soluble hIR{alpha}. In turn, the absolute amount of plasma insulin sequestered by hIR{alpha} in diabetic patients seemed to be much larger than that in normoglycemic subjects. In cases of the binding of insulin by insulin autoantibody, the antibody first sequesters insulin and then releases insulin after a time (15). Therefore, the apparent insulin-bound hIR{alpha} fraction may be underestimated. In addition, although we expected released soluble IR to participate in insulin resistance as one of the factors that contribute to glucose toxicity by sequestering plasma insulin, we found no correlations between hIR{alpha} level and other parameters reflecting insulin resistance (i.e., homeostasis model assessment of insulin resistance) or fasting IRI level in the present study.

According to clinical data (Fig. 2C and E), hIR{alpha} level changed more rapidly and dynamically than A1C or glycoalbumin levels. Therefore, we estimated the half-life of hIR{alpha} using STZ-induced diabetic TG mice (Fig. 3E). The half-life was estimated to ~6 h and was much shorter than that of A1C (30 days) or glycoalbumin (~17 days), suggesting that IR{alpha} could be a more rapid glycemic marker. We examined the daily profile of hIR{alpha} levels and observed little change throughout the day (online appendix Fig. 1F) in normoglycemic subjects. However, considering such a short half-life, it may be possible to observe daily change in patients with diabetes, especially those with brittle diabetes. Furthermore, the shedding of IR appeared to depend on biological response to hyperglycemia, while A1C or glycoalbumin are elevated in response to hyperglycemia simply by nonenzymatic mechanisms. Thus, plasma hIR{alpha} levels may reflect biological response to hyperglycemia.

Many membrane proteins have soluble ectodomains that are subject to proteolytic release, i.e., the process known as shedding (16). In most cases, shedding is caused via proteolytic cleavage by members of the ADAM (a disintegrin and metalloprotease) family of membrane-tethered zinc metalloproteinases (MMPs) (16). We have also observed suppressive effects of a general MMP inhibitor (GM-6001) on IR{alpha} shedding (T. Obata, K. Yokoyama, E. Okamoto, Y. Ebina, unpublished observations), suggesting the involvement of MMPs in this process. Most MMPs have COOH-terminal cytoplasmic tails that have been shown to be phosphorylated by various protein kinases including protein kinase C (PKC) (17,18). This phosphorylation subsequently activates protease activity (17,18). Under high-glucose conditions, the activation of PKC by de novo synthesis of diacylglycerol is well known (19,20), including in endothelial cells (21), which also express IR (22). All these data suggest a vicious cycle for glucose toxicity; high glucose stimulates PKC activation, which in turn activates MMPs that cause the release of soluble IR into the plasma, which sequesters insulin, thereby further raising glucose levels. The hypothesis regarding the vicious cycle is supported by our prior studies showing that the injection of purified hIR{alpha} into mice with elevated blood glucose levels in vivo (8). At this time, the source organ of soluble IR is unclear. Of note, although we detected plasma soluble IR in hIR-TG mice, the expression level of IR in the liver was extremely lower than that in other organs.

Regarding the shedding of other membrane proteins in the diabetic state, Lim et al. (23) reported that soluble CD40 ligand and soluble P-selectin levels were increased in plasma from patients with diabetes, and they proposed that the increments promoted atherothrombotic complications in cardiovascular disease. In the diabetic condition, other receptor proteins as well as IR are possibly shed from the cell membrane. However, the shedding of these membrane proteins, except IR, was not associated with glycemic control. In our study, we measured plasma soluble TNF-{alpha}R1 and soluble IL-6R levels and compared them with plasma hIR{alpha} levels (online appendix Fig. 4A and B). Plasma soluble TNF{alpha}-R1, but not soluble IL-6R, levels significantly correlated with hIR{alpha} levels (P < 0.001), suggesting that there may exist both common and distinct mechanisms between the shedding of IR and these receptors. Thus, the possible existence of various mechanisms for shedding needs to be considered.

In general, the physiological roles of ectodomain shedding of membrane receptors were diverse and complex. In the case of growth hormone receptor (GHR), the shedding of GHR generates growth hormone binding proteins that interact with growth hormone with high affinity and therefore downregulate the availability of the ligand (24). As in the case of soluble GHR, the majority of soluble receptor ectodomains appeared to inhibit the actions of their ligands (25), but soluble IL-6 receptor seems to act agonistically upon IL-6 binding (26). In contrast, in the case of TrkA (the receptor for nerve growth factor), residual membrane-associated fragments were found to be phosphorylated after ectodomain shedding, associated with intracellular signaling molecules, potentiating its action (27). In this case, the process appeared to play a compensative positive role. In fact, expression of ectodomain-truncated IR has also been shown to exhibit ligand-independent activation (28). At present, the physiological role of the process of IR ectodomain shedding remains unclear. Even in normoglycemic subjects, some basal hIR{alpha} was detected. Further study is necessary to clarify this issue.


   APPENDIX
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX
REFERENCES
 
Members of the Soluble Insulin Receptor Study Group.
Toshiyuki Obata,1 Ichiro Yokota,2 Kazuhiro Yokoyama,1 Eiji Okamoto,3 Yoshiko Kanezaki,1 Yoshinori Tanaka,1 Hiroshi Maegawa,4 Kiyoshi Teshigawara,1 Fumiko Hirota,5 Tomoyuki Yuasa,1 Kazuhiro Kishi,1 Atsushi Hattori,1 Seiichi Hashida,8 Kazuhiko Masuda,6 Mitsuru Matsumoto,5 Toshio Matsumoto,7 Atsunori Kashiwagi,4 and Yousuke Ebina.1

From the 1Division of Molecular Genetics, 5Division of Molecular Immunology, Institute for Enzyme Research, 2Department of Pediatrics, 7Department of Medicine and Bioregulatory Science, Graduate School of Medicine, The University of Tokushima, Tokushima, Japan; 3Medical & Biological Laboratories Co. Ltd., Nagoya, Japan; the 4Department of Medicine, Shiga University of Medical Science, Otsu, Shiga, Japan; 6Naruto Hospital, Naruto, Tokushima, Japan; and the 8Division of Life Style Diseases, Institutes for Health Science, Tokushima Bunri University, Tokushima, Japan.

T.O. and I.Y. contributed equally to this study. T.O. is currently affiliated with the Department of Medicine, Shiga University of Medical Science, Otsu, Shiga, Japan.


   ACKNOWLEDGMENTS
 
T.O., K.K., and Y.E. are supported by research grants from the Ministry of Education, Science, Technology, Sports and Culture of Japan. T.O. is supported by research grants from the 21st Centers of Excellence program of the University of Tokushima, the Insulin Research Foundation of Novo Nordisk Pharma, the Uehara Memorial Foundation, the Mitsui Social Welfare Foundation, the Japan Diabetes Foundation, and the Takeda Science Foundation.

The authors thank Dr. R. Roth (Stanford University, Stanford, CA) for anti-IR{alpha} antibody (5D9), K. Wakamatsu, M. Yamanaka, and C. Takehara for technical and secretarial assistance, and Dr. Y. Shintani (University of Tokushima, Tokushima, Japan) for assistance in blood sampling. We also thank Dr. J. Miyazaki (Osaka University, Osaka, Japan) for pCAGGS expression vector for TG mice.


   FOOTNOTES
 
* A complete list of the members of the Soluble Insulin Receptor Study Group can be found in the APPENDIX. Back

Published ahead of print at http://diabetes.diabetesjournals.org on 11 June 2007. DOI: 10.2337/db07-0394.

Additional information for this article can be found in an online appendix at http://dx.doi.org/10.2337/db07-0394.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received for publication March 23, 2007 and accepted in revised form May 15, 2007


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 DISCUSSION
 APPENDIX
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