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Endocrinology Vol. 139, No. 1 44-50
Copyright © 1998 by The Endocrine Society

ARTICLES

Effect of Glucose on Insulin-Like Growth Factor Binding Protein-4 Proteolysis1

Terry A. Jacot and David R. Clemmons

Renal Cell Biology Section, National Institute of Diabetes, Digestive, and Kidney Diseases, National Institutes of Health (T.A.J.), Bethesda, Maryland 20892; and Department of Medicine, University of North Carolina School of Medicine (D.R.C.), Chapel Hill, North Carolina 27599

Address all correspondence and requests for reprints to: David R. Clemmons, Division of Endocrinology and Metabolism, Department of Medicine, Campus Box 7170, 6111 Thurston-Bowles Building, University of North Carolina, Chapel Hill, North Carolina 27599-7170.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The role of hyperglycemia in diabetic changes of the insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) has not been clearly established. We therefore determined whether glucose modulates IGFBP synthesis and stability in vitro. Porcine vascular smooth muscle cells (pSMC) cultured in low glucose (pSMC-L) had 2.1-fold more IGFBP-4 in the conditioned medium compared with pSMC cultured in high glucose (pSMC-H) (P < 0.01). In contrast, IGFBP-2 levels remained constant. Although pSMC-H and pSMC-L cultures expressed similar levels of IGFBP-4 messenger RNA, in vitro protease assays demonstrated an increase in IGFBP-4 proteolysis in pSMC-H conditioned medium compared with pSMC-L conditioned medium (P < 0.01). The protease had properties similar to a previously characterized IGFBP-4 protease. The addition of 20 mM mannitol to pSMC-L cultures did not decrease IGFBP-4 levels, suggesting that the difference in IGFBP-4 proteolysis was not osmotically induced. The change was not due to selection bias, because cultures that were initially isolated from aortic explants in high and low glucose still exhibited the glucose-dependent difference in IGFBP-4 proteolytic activity. The results suggest that high glucose acts on pSMC to induce a change in IGFBP-4 proteolytic activity, which results in increased IGF-I availability to its receptors thereby enhancing the SMC proliferative response.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INSULIN-LIKE growth factor I (IGF-I) and its binding proteins (IGFBPs) have been shown to regulate the actions of IGF-I in the cardiovascular system (1, 2). Vascular smooth muscle cells (VSMC), an integral component of blood vessel walls, secrete IGF-I (3, 4) and have IGF-I receptors (5). IGF-I is also a important progression factor for VSMC proliferation (6). More recent studies determined that IGF-I has the ability to regulate fibronectin (7) and elastin (8) production by VSMC. Because IGF-I has a role in VSMC proliferation and matrix production, its possible involvement in diabetes- associated atherosclerosis is of great interest. Following arterial injury, increased aortic IGF-I messenger RNA (mRNA) has been demonstrated (9, 10). Atherosclerotic plaques also showed increased IGF-I by immunostaining (11). Although balloon injury to vessels in animals has consistently elicited IGF-I responses, the mechanisms by which IGF-I acts on blood vessel wall cell types in diabetic animals is less well defined. The DNA synthesis response of vessel wall cell types to systemically administered IGF-I is decreased in diabetic rats compared with normal rats (10). However, the response to locally produced IGF-I has not been assessed. Studies have shown that IGF-I expression is enhanced in macrophages in response to advanced glycation products (12), and macrophages may be an important local source of IGF-I within lesions. Furthermore, IGF-I receptor number is increased in endothelial (13) and mesangial (14) cells from diabetic animals.

IGFBPs have a very high affinity for IGF-I and IGF-II and can regulate IGF access to cell surface receptors. Therefore, there has been a great deal of interest in the alterations of these proteins that may occur in diabetes. IGFBP-1 in plasma is increased (15, 16), whereas IGFBP-3 (17, 18) is decreased in both diabetic rats and humans. The tissue expression patterns of IGFBP-1 and IGFBP-3 are distinct. IGFBP-1 mRNA is increased in rat livers and kidneys (19, 20), whereas IGFBP-3 mRNA is decreased in these tissues (21). Although these studies describe the effect of diabetes on IGFBP synthesis and/or steady state mRNA levels, whether other processes such as posttranslational modification of IGFBPs occur in the hyperglycemic state are unknown.

We have previously demonstrated IGFBP-2, IGFBP-4, and IGFBP-5 secretion by porcine aortic smooth muscle cells (pSMC) in vitro (22, 23). Because IGFBPs are important modulators of IGF actions in VSMC, we determined whether hyperglycemia altered IGFBP mRNA abundance or IGFBP peptide stability.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
DMEM, penicillin, streptomycin, and FBS were purchased from GIBCO/BRL (Grand Island, NY). Tissue culture dishes were obtained from Costar (Cambridge, MA). Glutamine, mannitol, EDTA, and phyenylmethylsulfonylfluoride (PMSF) were purchased from Sigma (St. Louis, MO). 35S-labeled L-methionine (1152 Ci/mmol) was purchased from ICN (Irvine, CA). PVDF membranes were from Millipore (Bedford, MA). 125I-labeled IGF-I (2000 Ci/mmol), 32P-labeled deoxycytidine triphosphate (3000 Ci/mmol) were obtained from Amersham (Arlington Heights, IL). Immunoblotting was performed using reagents that were purchased from Amersham or Tropix (Bedford, MA). Human IGFBP-4, bovine IGFBP-2, and their antisera were prepared as previously described (24, 25). IGF-II was a gift from Monsanto (St. Louis, MO), whereas IGF-I was purchased from GIBCO/BRL.

Tissue culture
Porcine aortic smooth muscle cells (pSMC) were isolated from thoracic aortas of 3-week-old piglets (26) and maintained in DMEM supplemented with 10% FCS, 4.5 g/liter glucose (25 mM), 4 mM glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin (DMEM-H). These cells were designated as pSMC-H. Some pSMC-H were transferred and cultured in DMEM supplemented with the same components as above, except that it contained 1.0 g/liter (5 mM) glucose (DMEM-L). These cells were designated as pSMC-L. For other experiments, pSMC aortic explants were directly cultured in either DMEM-H or DMEM-L. These primary cultures were labeled pSMC-H1 and pSMC-L1. For secretion experiments, cells were plated a density of 1.0 x 104 cells/cm2 in 24-well culture dishes. After 6 days, serum-free DMEM-H or DMEM-L supplemented with 0.1 mg/ml BSA was added to the cultures for 24 h, after which the conditioned medium was collected and stored for future IGFBP-2 and -4 analysis. Cells were then counted using a Coulter counter (Hialeah, FL). For the reversal experiments, pSMC-L cultures in 24-well dishes were grown to the same density as previously stated and then switched into the high-glucose medium 1, 3, or 6 days after initial plating. All cultures were maintained for a total of 6 days in serum-containing medium and 1 day in serum-free medium plus BSA. At that time, conditioned medium was collected and analyzed for IGFBP-4 by ligand- or immunoblotting. To control for the effect of changes in osmolarity, pSMC-L were plated as described above and grown in DMEM-L ± 20 mM mannitol for 6 days. The cultures were then changed into serum-free DMEM-L ± mannitol for 24 h. Conditioned media were collected and stored for IGFBP-4 analysis. Cells between passage 6 and 12 were used.

Total protein synthesis and secretion
pSMC-H and pSMC-L were plated in 6-well culture dishes at a density of 1.0 x 104 cells/cm2, grown to confluency, and incubated for 24 h in serum-free DMEM-H or DMEM-L. The cells were then preincubated in 1 ml methionine-free medium at 37 C for 45 min and 15 µC [35S]L-methionine was added for 6 h at 37 C. The conditioned media were collected and placed on ice. The cells were rinsed twice with cold PBS/0.1% BSA, and the rinses pooled with the conditioned media. The cells were scraped from the plate in cold 5% trichloracetic acid (TCA), the wells washed twice with 5% TCA, and the washes pooled with the cellular fractions. These cellular lysates were sonicated for 5 min and then precipitated at 4 C. After centrifugation for 10 min at 16,000 x g, the pellets were washed three times with cold 5% TCA and resuspended in 0.1 N NaOH. Scintillation fluid was added, and the samples counted.

To quantify the synthesis of proteins secreted into the media, the conditioned media was pooled with two PBS rinses and then centrifuged for 5 min at 16,000 x g to remove cellular debris. The supernatants were collected, TCA precipitated, washed, and counted as described above.

Ligand blotting
Binding proteins in pSMC conditioned media were separated in 12.5% nonreducing SDS-polyacrylamide gels and subsequently transferred to polyvinylidene difluoride membranes. For IGFBP-2 analysis, 21 µl conditioned medium was electrophoresed. To analyze the less abundant IGFBP-4, 75 µl conditioned medium was lyophilized, resuspended in 30 µl loading buffer, and then electrophoresed. If the differences in final cell densities between the cell types were greater than 10%, loading volumes were corrected for cell number. The membranes were sequentially blocked in TBS with 3% Nonidet P-40, TBS with 1% BSA, and TBS with 0.1% Tween-20 before an overnight incubation at 4 C with 125I-labeled IGF-I at a concentration of 100,000–200,000 cpm/ml in TBS containing 1% BSA, 0.1% Tween-20, pH 7.4. The membranes were washed and exposed to film for 3–6 days. The band intensity was quantified using scanning densitometry.

In vitro protease assays
Purified human IGFBP-4 or IGFBP-2 was added to 35 µl conditioned media of pSMC-H and pSMC-L cultures at final concentrations of 1 µg/ml or 500 ng/ml, respectively, and incubated in the absence or presence of 50 ng/ml IGF-I or IGF-II at 37 C for various times ranging between 2–24 h. Some samples were concentrated 4-fold to optimize degradation of the exogenous IGFBP-4 and to obtain more accurate scanning densitometric measurements. The reaction was then terminated by the addition of an appropriate amount of 4x Laemmli sample buffer and half of the reaction mixture applied to a 12.5% nonreducing SDS-polyacrylamide gel. After transfer to a PVDF membrane, intact IGFBP-4 or IGFBP-2 and its fragments were detected by immunoblotting. For IGFBP-4 detection, the membranes were blocked in 5% nonfat dry milk in TBS plus 0.1% Tween-20 followed by an overnight incubation in the blocking buffer containing a 1:4000 dilution of rabbit antihuman IGFBP-4 antiserum. After three washes in TBS plus 0.1% Tween 20, the membranes were then incubated with a 1:40,000 dilution of a goat antirabbit IgG-conjugated horseradish peroxidase for 1 h. After five washes, the bound antibodies were detected using the enhanced chemiluminescence substrate according to the protocol provided by Amersham. Intact IGFBP-4 was quantified using scanning densitometry. IGFBP-2-containing membranes were also blocked in the same blocking buffer followed by an overnight incubation with 1:6000 dilution of a rabbit antibovine IGFBP-2 antiserum. After washing, the membranes were incubated with a 1:20,000 goat antirabbit IgG-conjugated alkaline phosphatase for 1 h. After three washes, bound antibodies were detected using 1, 2-dioxetane alkaline phosphatase substrate (Tropix) according to the protocol supplied by the manufacturer.

To determine the effects of protease inhibitors on IGFBP-4 proteolysis, final concentrations of 10 mM EDTA, 10 mM PMSF, or 1 mM 3,4 dichloroisocoumarin (3,4 DCI) was added to pSMC-L and pSMC-H conditioned media containing exogenously added IGFBP-4 (1 µg/ml). The samples were then incubated in the presence or absence of 50 ng/ml IGF-II at 37 C for 24 h, and the remaining intact IGFBP-4 detected by immunoblotting as described above. To determine whether the proteolytic activity was increased by direct exposure to glucose, 20 mM glucose was added to pSMC-L conditioned media containing exogenously added IGFBP-4 and IGF-II. The samples were then incubated at 37 C for 24 h and intact IGFBP-4 detected as described above.

Northern blotting
pSMC-L and pSMC-H were grown in 25 cm2 flasks to a density of 4.0 x 105 cells. RNA was extracted using a previously described SDS-acid phenol/chloroform method (27). Cells were lysed in 1% SDS and 10 mM EDTA, pH 8.0, followed by the addition of a solution containing 0.1 M sodium acetate, pH 4.0, and 10 mM EDTA. Buffered phenol, pH 4.3, was added to the samples, which were vortexed and incubated on ice for 15 min. Samples were centrifuged for 10 min at 4 C, and the aqueous phase collected and re-extracted with an equal volume of a 5:1 phenol-chloroform/isoamyl alcohol (24:1) solution. After repeated centrifugation, RNA in the aqueous phase was precipitated with 5 M NaCl and 100% ethanol. Total RNA (10 µg) was separated in a 1% formaldehyde agarose gel and transferred to a nylon membrane by capillary action. Ethidium bromide staining was employed to control for equal loading. The membrane was hybridized with a 32P-labeled IGFBP-4 complementary DNA in 0.25 M Na2HPO4 containing 7% SDS at 65 C for approximately 16–18 h (28). The membrane was then washed two times for 30 min at 65 C in 0.04 M sodium phosphate plus 5% SDS and one time for 30 min at 65 C in 0.04 M sodium phosphate plus 1% SDS. The membrane was exposed to film for 4–6 days.

Quantitation of Northern, ligand, and Western blots
Northern, ligand, and Western blots were quantitated through scanning densitometry using a Hoefer GS-300 Scanning Densitometer (San Francisco, CA) or NIH Image, Version 1.6. Values were analyzed via Student’s unpaired t test and one-way ANOVA using Instat, Version 2.0. For nonnormally distributed data, the values were analyzed by the Mann-Whitney test. Data represents at least three independent experiments. Scanning data are expressed as the mean ± SEM.

Measurement of IGF-II
IGF-II concentrations were measured in pSMC-L and pSMC-H serum-free media collected after 24 h as described previously by a specific RIA after removal of IGFBPs using a previously described method (29).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of glucose concentration on IGFBP-4 in conditioned medium
Endogenous IGFBP-4 levels in conditioned media from pSMC cultured in 25 mM glucose (pSMC-H) and pSMC-H switched into 5 mM glucose (pSMC-L) were analyzed by ligand blotting. IGFBP-4 levels in the conditioned media from pSMC-L cultures were 2.1-fold higher compared with pSMC-H cultures (P < 0.01) (Fig. 1Go and Table 1Go). IGFBP-2 levels were also quantified to assess whether different glucose conditions affected this binding protein. Comparison of IGFBP-2 from high or low glucose-exposed cultures showed no difference, implying a specific effect on IGFBP-4 levels by glucose. To exclude the possibility that glucose was inducing a change in total protein synthesis, this parameter was analyzed in the media and cell lysates. There was no difference between the two culture conditions, thereby confirming that the change in IGFBP-4 levels did not reflect a change in total protein secretion (pSMC-H media, 103,589 ± 5,672 cpm vs. pSMC-L media, 108,858 ± 14,418 cpm, P = NS; pSMC-H cell lysate, 372,539 ± 3,790 cpm vs. pSMC-L cell lysate, 36,7245 ± 2,182 cpm, P = NS). IGFBP-4 levels appeared to be quite sensitive to changes in glucose. Switching cultures from low-glucose to high-glucose media 1, 3, or 6 days after plating demonstrated that the effect of high glucose on IGFBP-4 was present within 24 h, and additional exposure time was not required (Fig. 2Go). Mannitol at a final concentration of 20 mM was added to pSMC-L cultures to control for changes in osmolarity. Under these conditions, no decrease in IGFBP-4 was observed, thereby confirming that the effect of glucose on IGFBP-4 was not due to an osmotically induced change in the culture media (Fig. 3Go).



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  		Figure 1. Effect of glucose on IGFBP-2 and -4 levels in pSMC-conditioned media. A representative ligand blot of 21 µl conditioned media (lanes 1–4) or 75 µl conditioned media (lanes 5–8). Both 34,000-kDa IGFBP-2 (upper arrow) and less abundant 24,000-kDa IGFBP-4 (lower arrow) were detected. Lanes 1, 3, 5, and 7 are conditioned media from cultures exposed to 25 mM glucose, whereas lanes 2, 4, 6, and 8 are conditioned media from 5 mM glucose-exposed cultures.

 

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  		Table 1. Effect of glucose on IGFBP-2 and -4 levels in conditioned media

 


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  		Figure 2. Effect of glucose reversal on endogenous IGFBP-4 levels in pSMC-L cultures. A representative ligand blot of concentrated conditioned media from pSMC-L cultures that had been switched into media containing high glucose 1 day (lanes 3 and 4), 3 days (lanes 5 and 6), or 6 days (lanes 7 and 8) after initial plating. Lanes 1 and 2 represent IGFBP-4 levels (lower arrow) after 7 days in low-glucose culture media as a control.

 


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  		Figure 3. Effect of mannitol on IGFBP-4 secretion by pSMC-L cultures. A representative ligand blot demonstrating IGFBP-4 levels (lower arrow) in conditioned media from pSMC-L cultures grown in presence (lanes 2 and 4) or absence (lanes 1 and 3) of 20 mM mannitol. After cells were grown with or without mannitol for 6 days and a 24-h incubation in serum- free media ± mannitol, conditioned media was collected, concentrated, and ligand blotted to determine endogenous IGFBP-4 levels.

 
To determine whether glucose was altering IGFBP-4 levels by altering its synthesis, total RNA from SMC-L and SMC-H cultures were analyzed by Northern blotting. There was no difference in the abundance of the 2.4-kb IGFBP-4 band intensity when the two glucose conditions were compared. Scanning densitometry of the IGFBP-4 band intensity in two separate experiments gave values of pSMC-H, 407 ± 94 vs. pSMC-L, 466 ± 41 OD units; Fig. 4Go).



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  		Figure 4. Effect of glucose on IGFBP-4 mRNA. A, Representative Northern blot demonstrating IGFBP-4 mRNA from pSMC-H cultures (lanes 1–4) and pSMC-L cultures (lanes 5–8). Cells were grown to confluency and exposed to appropriate serum-free media for 24 h. RNA was extracted from pSMC-H and pSMC-L cultures, and 10 µg total RNA analyzed. Ethidium bromide- stained gel is also shown. B, Scanning densitometry data of two independent Northern blots (n = 9). pSMC-H, 407 ± 94; pSMC-L, 466 ± 41

 
Effect of glucose on IGFBP-4 proteolysis
We have previously determined that pSMC medium contains a specific protease for IGFBP-4 (30). Because IGFBP-4 mRNA levels did not decrease in proportion to the decrease in this peptide found in pSMC-H cultures, we investigated whether IGFBP-4 proteolytic activity was affected by different glucose concentrations in the culture media. When cell-free conditioned media from pSMC-L and pSMC-H cultures were incubated with 1 µg/ml exogenously added human IGFBP-4 and 50 ng/ml IGF-II, fragments of approximately 19 and 14 kDa were detected by immunoblotting (Fig. 5Go). There was a time-dependent decrease of intact IGFBP-4 over the course of 24 h. At each time point, there was less intact IGFBP-4 in conditioned media than was obtained from pSMC-H cultures as compared with the media from pSMC-L cultures. To quantitate the IGF-II-induced proteolytic activity, changes in intact IGFBP-4 were measured by scanning densitometry. Although proteolytic fragments were detectable in both pSMC-L and pSMC-H conditioned media as compared with their own controls, e.g. no added IGF-II, IGFBP-4 proteolysis was significantly greater in the high-glucose media than in the low-glucose media (P < 0.01, Table 2Go). The effect appeared to be specific for IGFBP-4, because analysis of IGFBP-2 proteolytic activity showed no difference when media from pSMC-L and pSMC-H cultures were compared using the same conditions (data not shown).



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  		Figure 5. Comparison of IGFBP-4 proteolysis between pSMC-H and pSMC-L cultures. Exogenously added IGFBP-4 at a final concentration of 1 µg/ml was incubated with conditioned media from pSMC-H cultures (odd numbers) or pSMC-L cultures (even numbers) in presence of 50 ng/ml IGF-II for 2 h (lanes 1 and 2), 4 h (lanes 3 and 4), 6 h (lanes 5 and 6), 8 h (lanes 7 and 8), and 24 h (lanes 9 and 10). Samples without IGF-II (lanes 11 and 12) were also incubated for 24 h as controls. Intact IGFBP-4 (thick arrow) and its fragments (thin arrows), which were detected by immunoblotting, are shown.

 

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  		Table 2. Effect of glucose on IGFBP-4 proteolysis

 
Our previous studies have shown that the protease that degrades IGFBP-4 is a calcium-dependent serine protease (30). The proteolytic activity that was increased with glucose could be inhibited by both EDTA and PMSF (Fig. 6Go). A more specific serine protease inhibitor, 3,4 DCI, was also effective in inhibiting IGFBP-4 proteolysis (Fig. 6Go). These results suggest that glucose has some regulatory control on the serine protease that was characterized previously. To determine whether the change in IGFBP-4 proteolysis is a result of a direct interaction between glucose and the protease, 20 mM glucose was added to pSMC-L conditioned media containing exogenously added IGFBP-4 and IGF-II before performing incubation at 37 C for 24 h. Two independent experiments demonstrated no increase in proteolytic activity when extra glucose was added to the media. Therefore, the protease activity did not appear to be modified directly by glucose (data not shown). To exclude the possibility that high glucose was increasing IGFBP-4 protease activity through an increase in IGF-II, we measured the IGF-II concentration in the pSMC media. Triplicate determinations from two separate experiments showed that pSMC-H media contained 8 ± 3 ng/ml and pSMC-L media had a value of 7 ± 2 ng (P, NS). In a separate experiment, we noted that 5 ng/ml of IGF-II was a sufficient amount to detect IGFBP-4 proteolysis in vitro (data not shown). Therefore, we conclude that high glucose is not stimulating IGFBP-4 proteolytic activity by stimulating IGF-II. In summary, the increase in IGFBP-4 proteolysis seen in conditioned media from pSMC-H cultures is most likely a result of an increase in the amount of the IGFBP-4 protease or to a decreased amount of an inhibitor.



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  		Figure 6. Effect of protease inhibitors on IGFBP-4 proteolysis. A representative immunoblot showing inhibition of IGFBP-4 proteolysis by various inhibitors. IGFBP-4 (1 µg/ml) and IGF-II (50 ng/ml) were added to conditioned media obtained from pSMC cultures grown in low (even numbers) or high (odd numbers) glucose-containing media. Before 24 h incubation at 37 C, 10 mM PMSF (lanes 5 and 6) 10 mM EDTA (lanes 7 and 8), or 1 mM 3,4 DCI (lanes 9 and 10) were added to samples. Intact IGFBP-4 (thick arrow) and its fragment (thin arrows) were then detected by immunoblotting. Inhibition was compared with those samples without protease inhibitors (lanes 3 and 4). Samples incubated without IGF-II were used as controls (lanes 1 and 2).

 
IGFBP secretion by pSMC originally isolated in high or low glucose
To address the possibility that the difference between pSMC-H and pSMC-L might reflect a selection bias that occurred during cellular outgrowth from explants in medium containing 25 mM glucose, new cells were isolated from explants placed in DMEM containing either 5 mM or 25 mM glucose. The cells were designated as pSMC- L1 and pSMC- H1. Cultures grown in either glucose concentration were otherwise maintained exactly as in the previous experiments, and IGFBP-4 levels were compared. Ligand blotting showed that IGFBP-4 levels in pSMC-L1 cultures were higher than the levels found in pSMC-H1 cultures (Fig. 7Go). This observation confirms that the difference in IGFBP-4 proteolysis being reported is dependent on glucose and not a characteristic of phenotypically altered cells that had been isolated using high-glucose-containing medium. In contrast to IGFBP-4, IGFBP-2 levels in pSMC-L1 cultures were increased compared with pSMC-H1 cultures, a finding that was not observed when cells were changed from high glucose to low glucose. This increase in IGFBP-2 is most likely due to a change in IGFBP-2 synthesis, because the in vitro proteolysis assay demonstrated no change in IGFBP-2 degradation (data not shown).



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  		Figure 7. IGFBP-4 Secretion by pSMC-H1 and pSMC-L1 cultures. pSMC were cultured from aortic explants in either high or low glucose. A representative ligand blot of 100 µl conditioned media from pSMC-H1 (lanes 1 and 2) and pSMC-L1 (lanes 3 and 4) cultures. A, One-day exposure demonstrating increased IGFBP-2 levels in pSMC-L1 cultures. B, Same ligand blot exposed for 3 days to demonstrate increased IGFBP-4 levels in pSMC-L1.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
IGFBPs are important modulators of IGF activity. pSMCs have been shown to synthesize and secrete IGFBP-2, IGFBP-4, and IGFBP-5 (22, 23). Although IGFBP-2 and IGFBP-5 have inhibitory as well as stimulatory actions (31, 32), IGFBP-4 has been shown to consistently inhibit IGF-I actions (23, 33, 34, 35). The present experiments demonstrate that pSMCs cultured in high glucose have lower IGFBP-4 levels compared with pSMCs cultured in low glucose. The difference was not due to cell selection because it was detected even if the cells were initially isolated in low- or high-glucose medium. Although there was no difference in IGFBP-4 mRNA levels between low- and high-glucose cultures, glucose did appear to induce an increase in IGFBP-4 proteolysis, because an in vitro protease assay using exogenously added IGFBP-4 and IGF-II demonstrated less intact IGFBP-4 in pSMC-H-conditioned media as compared with pSMC-L-conditioned media.

We have previously reported decreased IGFBP-4 in pSMC cultures following exposure to IGF-I or II (23), and have attributed this change to an IGF-activated IGFBP-4 protease in the conditioned medium of these cultures (30). The IGFBP-4 proteolytic activity that was released by pSMC-H cultures showed: 1) enhanced activity following IGF-II addition, 2) inhibition by EDTA, 3,4 DCI, and PMSF, and 3) degradation into fragments of similar size to those previously reported. Taken together, these data strongly suggest that high glucose is stimulating the cells to release the same protease that we and others (36) have reported previously to be produced by smooth muscle cells.

The mechanism by which glucose induces a change in IGFBP-4 proteolysis was only partially addressed by these studies. The protease in pSMC-L media was not further activated by direct exposure to 20 mM glucose. In addition, high glucose did not appear to induce a change in IGF-II concentrations. Therefore, we conclude that glucose is stimulating the pSMC-H cultures to release more protease than the pSMC-L cultures without directly altering protease activity in vitro. The necessity to add IGF-II to the in vitro protease assay raises the question as to whether this mechanism fully accounts for the difference in intact IGFBP-4 noted when pSMC-L and pSMC-H cultures were compared. Several observations suggest that this conclusion is valid. The level of IGF-II released by the cultures (e.g. 8 ng/ml) was sufficient to detect proteolysis in IGFBP-4 in vitro. There are several differences between the in vivo and in vitro protease assay systems. In vivo, the cells continue to release protease throughout the 24-h incubation period, whereas in the in vitro assay, the amount is fixed. The cells, in vivo, also continue to release IGF-II. Therefore, the in vitro protease assay may require greater amounts of added IGF-II to detect an effect. In summary, we conclude that high glucose concentrations are modulating IGFBP-4 proteolysis through an effect on protease levels in our pSMC cultures.

Possible mechanisms by which glucose might function include increased synthesis and secretion of the protease, increased conversion from an apoenzyme to an active form, or decreased synthesis of a protease inhibitor. Two of these mechanisms have been proposed to regulate IGFBP-4 proteolysis in other systems. It has been proposed that glucocorticoid stimulates the secretion of an IGFBP-4 protease by a rat neuronal cell line (37). In contrast, Conover et al. (38) showed that phorbol ester treatment of human fibroblasts resulted in increased secretion of an IGFBP-4 protease inhibitor. The current literature therefore supports our proposal that glucose could directly alter the expression of a protease or a protease inhibitor.

The effect of glucose also appears to be specific for IGFBP-4 proteolysis. Previous studies have shown that pSMCs also produce a proteolytic activity for IGFBP-2 (39) and IGFBP-5 (22). In these experiments, there was no difference in IGFBP-2 levels or proteolytic activity in conditioned medium obtained from pSMC-H and pSMC-L cultures. In addition, the IGFBP-4 effect did not reflect general changes in total protein synthesis because high- and low-glucose pSMC cultures had similar amounts of [35S]methionine-labeled cellular and/or secreted proteins. The lack of a difference is not too surprising because protein synthesis was measured after a 24-h incubation in medium lacking serum. The effect was also not due to differences in cell number, because there was an average difference of only 10% in final cell number. Although others (36) have postulated that IGFBP-4 proteolytic activity released by rat smooth muscle cells in culture is confluency dependent, the changes that were reported resulted from greater than 3-fold differences in cell number, and therefore a 10% difference in cell number would be unlikely to account for the large difference (e.g. a 114% increase) in IGFBP-4 levels that we observed. Further evidence against the effect being due to changes in cell number are the reversal experiments shown in Fig. 3Go. If the differences in IGFBP-4 were due to differences in the rate of cell growth, one would expect to see a time-dependent change in IGFBP-4 levels in the cultures that were switched to the high-glucose medium. However, glucose reversal induced essentially the same effect whether the pSMC-L cultures were maintained in high glucose for 1, 3, or 6 days. Taken together, these findings suggest that the effect of glucose is specific for altering the IGFBP-4 specific protease.

To exclude the possibility that high glucose induced a phenotypic change, or that it selectively permitted survival of a phenotypically distinct subpopulation of cells, freshly isolated aortic explants were cultured in DMEM with low or high glucose. IGFBP-4 levels in the pSMC cultures containing low glucose were again higher than IGFBP-4 levels in pSMC cultures containing high glucose. However, unlike the pSMCs that were switched from high to low glucose, the cultures containing cells that had been isolated in low-glucose medium had higher levels of IGFBP-2. In vitro protease assays for IGFBP-2 protease activity did not show any change in IGFBP-2 proteolysis, so the difference is most likely due to a change in synthesis rather than stability. These results confirm that the difference in IGFBP-4 levels is a result of a glucose-associated change in protease activity rather than a hyperglycemia-induced phenotypic alteration in the subpopulation of pSMCs that was being analyzed. Although these new pSMC cultures show that IGFBP-2 levels may reflect possible phenotypic changes in response to different glucose conditions, they strengthen the conclusion that the glucose effect on IGFBP-4 proteolysis is not the result of a phenotypic change or a cell selection phenomenon.

In contrast to glucose, insulin increases IGFBP-4 synthesis by pSMC. Insulin results in a 2-fold increase in IGFBP-4 mRNA and a substantial increase in detectable protein in conditioned medium (23). The insulin-induced effect could function in concert with the glucose-induced change, because IGFBP-4 proteolysis in the absence of insulin would be associated with a decrease in IGFBP-4 synthesis and an increase in its rate of disappearance. Conversely, in states of insulin sufficiency, one would expect to see increased IGFBP-4 synthesis and decreased degradation due to lowering of glucose concentrations.

IGF-I has been implicated as a potentially important mediator of atherosclerotic lesion formation because IGF-I gene expression is increased in the aorta after balloon injury. Macrophages that are activated by exposure to advanced end glycosylation products also secrete increased amounts of IGF-I. When PDGF is released from platelets at the site of injury, it also enhances IGF-I synthesis. Although our findings do not prove a causal link between degradation of IGFBP-4 and SMC proliferation, IGFBP-4 consistently inhibits IGF actions including smooth muscle cell proliferation. Therefore, the findings suggest that glucose regulation of IGFBP-4 proteolysis represents a potentially important mechanism for diminishing the inhibitory effect of the binding protein on IGF-I action in the atherosclerotic lesion microenvironment. Future studies directed toward determining the role of this protease in vivo should help to determine its relevance to the response of smooth muscle cells in vivo to the hyperglycemic progression of atherosclerosis.


   Acknowledgments
 
We thank Drs. Liliane and Gary Striker for the use of their laboratory resources without which this study could not have been completed. We thank Dr. Louis Underwood (University of North Carolina) for performing the IGF-II RIA.


   Footnotes
 
1 This work was supported by a grant from the National Institutes of Health (HL-56580). Back

Received May 1, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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