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Originally published In Press as doi:10.1074/jbc.M502927200 on June 21, 2005

J. Biol. Chem., Vol. 280, Issue 33, 29946-29955, August 19, 2005
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Accelerated Glucose Intolerance, Nephropathy, and Atherosclerosis in Prostaglandin D2 Synthase Knock-out Mice*

Louis Ragolia{ddagger}§, Thomas Palaia{ddagger}, Christopher E. Hall{ddagger}, John K. Maesaka{ddagger}§, Naomi Eguchi||**, and Yoshihiro Urade||

From the {ddagger}Vascular Biology Laboratory, Winthrop-University Hospital, Mineola, New York 11501, §Stony Brook University School of Medicine, Stony Brook, New York 11794, and the ||Osaka Bioscience Institute, Osaka, Japan

Received for publication, March 16, 2005 , and in revised form, June 21, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Type 2 diabetics have an increased risk of developing atherosclerosis, suggesting the mechanisms that cause this disease are enhanced by insulin resistance. In this study we examined the effects of gene knock-out (KO) of lipocalin-type prostaglandin D2 synthase (L-PGDS), a protein found at elevated levels in type 2 diabetics, on diet-induced glucose tolerance and atherosclerosis. Our results show that L-PGDS KO mice become glucose-in-tolerant and insulin-resistant at an accelerated rate when compared with the C57BL/6 control strain. Adipocytes were significantly larger in the L-PGDS KO mice compared with controls on the same diets. Cell culture data revealed significant differences between insulin-stimulated mitogen-activated protein kinase phosphatase-2, protein-tyrosine phosphatase-1D, and phosphorylated focal adhesion kinase expression levels in L-PGDS KO vascular smooth muscle cells and controls. In addition, only the L-PGDS KO mice developed nephropathy and an aortic thickening reminiscent to the early stages of atherosclerosis when fed a "diabetogenic" high fat diet. We conclude that L-PGDS plays an important role regulating insulin sensitivity and atherosclerosis in type 2 diabetes and may represent a novel model of insulin resistance, atherosclerosis, and diabetic nephropathy.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cardiovascular disease is the primary cause of morbidity and mortality in people with non-insulin-dependent diabetes mellitus (1). Type 2 diabetics have a significantly increased risk of developing hypertension, atherosclerosis, and restenosis after angioplasty or stent implantation (2, 3). These phenomena are partially attributable to the abnormal accumulation of vascular smooth muscle cells (VSMCs)1 within the intima of blood vessels resulting from alterations in migration, proliferation, and apoptosis (4, 5).

Previously, we have demonstrated that lipocalin-type prostaglandin D2 synthase (L-PGDS) inhibits the exaggerated growth phenotype of VSMCs isolated from hypertensive rats (6). The mechanism appears to involve the inhibition of insulin-stimulated protein kinase B (Akt) and glycogen synthase kinase-3{beta} phosphorylation (6). We have also shown that VSMC apoptosis and migration are both altered by L-PGDS and that any beneficial effects of L-PGDS are absent in VSMCs isolated from diabetic animals (7). In addition, L-PGDS is ultimately responsible for the synthesis of the naturally occurring peroxisome proliferator activator receptor {gamma} ligand, 15-deoxy-{Delta}12,14 prostaglandin J2, already known to induce apoptosis (8). Moreover, thiazolidinediones, which are synthetic peroxisome proliferator activator receptor {gamma} ligands, have been shown to increase insulin sensitivity and enhance insulin-stimulated glucose transport into muscle (9) and exert other beneficial cardiovascular effects such as blocking VSMC growth and migration to delay the onset of atherosclerosis (10, 11).

Several other findings have emerged suggesting that L-PGDS has important vascular functions. Inoue et al. (12) have demonstrated that elevated serum L-PGDS levels correlate with a decreased occurrence of restenosis after coronary angioplasty. A polymorphism in the L-PGDS gene appears to be associated with carotid atherosclerosis in a population of hypertensive Japanese individuals (13); the balance between L-PGDS and prostaglandin (PG) E synthase has been shown to be a major determinant of atherosclerotic plaque instability (14); laminar shear stress stimulates vascular endothelial cells to up-regulate L-PGDS expression (15); human L-PGDS mRNA expression has been measured to be highest in heart tissue, with enzyme immunoreactivity localized in myocardial cells, arterial endocardial cells, and the synthetic state of smooth muscle cells in arteriosclerotic plaques (16); chicken apolipoprotein D and L-PGDS share similar protein sequences (17); urinary L-PGDS excretion has been shown to increase in the early stages of diabetes mellitus (18). Collectively, these studies illustrate the need to clarify the role of L-PGDS in the vasculature, especially in atherosclerosis, hypertension, and diabetes.

The C57BL/6 mouse strain represents one model to study diet-induced atherosclerosis and diabetes (19). After 20 weeks on a high fat (42% kcal) diabetogenic diet the mice become hyperglycemic, insulin-resistant, and obese. Given our previous observations on the effects of L-PGDS and the vascular implications of L-PGDS, we decided to examine the effects of a high fat diet on insulin resistance and atherosclerosis in L-PGDS knock-out (KO) mice.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture reagents, including fetal bovine serum, were purchased from Invitrogen. SDS/polyacrylamide gel electrophoresis and Western blot reagents were from Bio-Rad. Antibodies to mitogen-activated protein kinase phosphatase (MKP)-2 and phosphotyrosine phosphatase-1D (PTP-1D) were purchased from BD Biosciences and used at dilutions of 1:500 and 1:2500, respectively. Focal adhesion kinase (FAK) and pFAK antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and used at a dilution of 1:200. Bicinchoninic acid protein assay reagent was purchased from Pierce. Western blots were visualized with enhanced chemiluminescence reagent purchased from Amersham Biosciences. Type-1 collagenase was from Worthington Biochemical Co. (Freehold, NJ). All other reagents were of reagent grade or better and purchased from the Sigma.



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  		FIG. 1.
Mouse intraperitoneal glucose tolerance tests. Control C57BL/6 (n = 12) and L-PGDS KO (n = 12) male mice at 6-8 weeks of age (A), after 20 weeks on a low fat diet (B) and after 20 weeks on a high fat diet (C), were fasted overnight. Blood was collected before (T0) and after intraperitoneal injection of glucose (2 g/kg body wt) at the indicated times, and serum glucose was measured. Data are expressed as the means ± S.E. Asterisks (*) represent a p < 0.01 when compared with corresponding control C57BL/6 mice.

 
Animals and Diets—Male L-PGDS transgenic KO C57BL/6 mice, obtained from the Osaka Bioscience Institute (Osaka, Japan) (20), and C57BL/6NHla mice, purchased from Hilltop Lab Animals, Inc. (Scottsdale, PA), were maintained in temperature-controlled rooms (22 °C) with a 12-h light/dark cycle and given free access to food and water. Mice were fed a pellet-form rodent chow diet (Harlan Teklad Test Diets #8604, Madison, WI) before the initiation of studies. At 6-8 weeks of age animals were switched to either a high fat diet providing 45% of the calories as fat (D12451 [GenBank] ) or the corresponding control diet (D12450B), with 10% of the calories as fat (Research Diets Inc., New Brunswick, NJ). The fat source for both diets was identical, and equal numbers of mice were fed the low and high fat diets for up to 20 weeks.



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  		FIG. 2.
Mouse intraperitoneal insulin tolerance tests. Control C57BL/6 (n = 12) and L-PGDS KO (n = 12) male mice at 6-8 weeks of age (A), after 20 weeks on a low fat diet (B) and after 20 weeks on a high fat diet (C), were fasted for 5 h. Blood was collected before (T0) and after intraperitoneal injection of insulin (1 units/kg body wt) at the indicated times, and serum glucose was measured. Data are expressed as the means ± S.E. Asterisks (*) represent a p < 0.01 when compared with corresponding control C57BL/6 mice.

 
Analytical Procedures—Mice were fasted for 5 h before drawing tail blood for all experiments except glucose tolerance tests, when 16 h fasts were implemented. Fresh blood was assayed for glucose using a Bio-Scanner 2000 (Polymer Technology Systems, Inc., Indianapolis, IN), and the concentration was expressed as mg/dl. Serum concentrations of insulin were determined using the ultrasensitive rat/mouse insulin (Crystal Chem, Inc., Downers Grove, IL) enzyme-linked immunosorbent assay kit. Adiponectin levels of all molecular weights were measured using an enzyme-linked immunosorbent assay kit (B-Bridge International, Inc., Sunnyvale, CA). Concentrations of both insulin and adiponectin were extrapolated from a curve of known mouse standards plotted against assay absorbances.



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  		FIG. 3.
Ten-minute intraperitoneal glucose pulse. L-PGDS KO male mice at 6-8 weeks of age (Initial) after 10 weeks on a low fat diet (Low fat) and after 10 weeks on a high fat diet (High fat) were fasted overnight. Blood was collected before (T0') and after intraperitoneal injection of glucose (2 g/kg body wt) at 10 min (T10'), and serum insulin was measured as described under "Materials and Methods." Data are expressed as the means ± S.E. (n = 8 for each condition).

 
Intraperitoneal Glucose Tolerance Test and Acute Insulin Response—Intraperitoneal glucose tolerance tests were performed in animals fasted for 16 h. Blood was collected before (0 min) and after intraperitoneal injection of glucose (2 g/kg body wt) at 15, 30, 60, 90, and 120 min. In a separate series of experiments, overnight fasted mice (both control and L-PGDS KO) had initial bloods collected (T0) followed by glucose injection (2 g/kg body wt intraperitoneal) and another blood collection after 10 min (T10).

Intraperitoneal Insulin Tolerance Test—An intraperitoneal insulin tolerance test was performed after animals were fasted for 5 h. Blood glucose levels were measured before the injection of insulin (1.0 units/kg body wt; Novolin®, Novo Nordisk Pharmaceuticals, Inc., Princeton, NJ) and at 15, 30, 60, 90, and 120 min after injection.

Cell Culture—VSMCs were isolated by collagenase digestion of the aortic media from mice as described previously (7). VSMCs prepared from these mice were not contaminated with fibroblasts or endothelial cells as evidenced by a greater than 99% positive immunostaining of smooth muscle {alpha}-actin with fluorescein isothiocyanate-conjugated {alpha}-actin antibody. Subcultures of VSMCs were maintained in {alpha}-minimum Eagle's medium containing 10% fetal bovine serum and 1% antibiotic/antimycotic. Cells were synchronized in G0 by incubating in serum-free medium for 24 h. Cells were grown to confluency and studied at passages 5-6 for all experiments.

Western Blotting—Cells were lysed as previously described. When phosphorylation was detected, phosphate-buffered saline and cell lysis buffer contained 2 mM sodium vanadate and 1 µM microcystin at 4 °C. Typically, 50 µg of protein was mixed with Laemmli sample buffer containing 0.1% bromphenol blue, 1.0 M NaH2PO4, pH 7.0, 50% glycerol, and 10% SDS, boiled for 5 min, and loaded on an SDS-polyacrylamide gel. The separated proteins were transferred to polyvinylidene difluoride membrane and probed with the proper primary antibodies followed by 1:2000 diluted secondary antibody and detection with enhanced chemiluminescence reagent and subsequent autoradiography. The intensity of the signal was quantitated by densitometric analysis of the autoradiograms and normalized against {beta}-actin or, in the case of phosphorylations, the non-phosphorylated form of the protein.

Serum Creatinine—A creatinine diagnostic kit (Thermo Electron, Louisville, CO) was used to determine serum creatinine based on the formation of an alkaline picrate product with absorption at 500 nm.

Tissue Acquisition and Staining—Aorta, kidney, and visceral adipose excised from epididymal fat pads were excised and processed as described (21). The tissue was immediately placed in a vial containing 4% phosphate-buffered formalin. After dehydration and paraffin embedding, 4 µm sections were processed for staining. Aorta cross-sections and adipose were stained with hematoxylin (0.4%) and eosin (0.5%). For whole vessel staining, a stock solution of Oil Red O (3.5 mg/ml isopropanol) was diluted 1:1 with water and incubated with vessel for 10 min, rinsed in isopropanol, and transferred to distilled water. Kidney sections were stained with either trichrome stain or immunohistochemically using the rabbit ABC staining system as per the manufacturer's instructions (Santa Cruz Biotechnology). After treatment, sections were washed with phosphate-buffered saline and counterstained with hematoxylin. Control slides were incubated with nonspecific primary anti-sera or in some cases without the primary antibody. In no case did control slides show a positive signal.

Digital Quantification—Digital images of whole aortas, aortic cross-sections, and adipocytes were captured into SigmaGel (Systat Software, Inc., Richmond CA). For Oil Red O staining, the area of stained aorta (pixels2) was divided by total vessel area (pixels2) to give the percent of vessel stained. Aortic wall area was calculated by "flooding" the entire media and reporting pixels2. The mean area of adipocytes was calculated by measuring the area of individual adipocytes (pixels2) from five randomly chosen fields.

Statistics—Data are reported as the means ± S.E. Statistical differences were determined by Student's t test or analysis of variance where appropriate using SigmaStat 3.0. p < 0.05 was accepted as statistically significant. The Institutional Animal Care and Use Committee of Winthrop-University Hospital approved this study.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Glucose and Insulin Tolerance in L-PGDS KO Mice—The effects of a high fat (diabetogenic) diet on glucose and insulin tolerance were measured in L-PGDS KO mice as well as the C57BL/6 control strain. As seen in Fig. 1A, both mouse strains show rather similar initial glucose profiles in response to an intraperitoneal glucose tolerance test, with the L-PGDS KO curve shifted slightly higher than the control. Interestingly, although the control C57BL/6 mice seem to display typical glucose tolerance after 20 weeks on a low fat diet, the L-PGDS KO mice have already developed impaired glucose tolerance (Fig. 1B). Both strains exhibit impaired glucose tolerance after 20 weeks on the high fat diet (Fig. 1C).

An analogous situation is observed with insulin tolerance. Although both strains seem responsive to intraperitoneal insulin injection initially (Fig. 2A), only the L-PGDS KO mice are significantly less responsive to insulin after 20 weeks on the low fat diet (Fig. 2B). Insulin resistance is observed in both strains after 20 weeks on the high fat diet (Fig. 2C).

Blood Glucose and Insulin Levels in Response to Glucose—To determine whether defective insulin secretion contributed to the glucose intolerance observed in the L-PGDS KO mice, insulin levels were measured in response to intraperitoneal glucose injections. As seen in Fig. 3, glucose injection increased insulin secretion in L-PGDS KO mice initially; however, after 10 weeks on either diet, both sets of mice were hyperinsulinemic and hardly responsive to glucose injection (Fig. 3).



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  		FIG. 4.
Oil Red O staining of mouse aorta. Control C57BL/6 (n = 9) and L-PGDS KO (n = 9) male mice were sacrificed after 20 weeks on a low fat or high fat diet. Aorta were removed and stained with Oil Red O as described under "Materials and Methods" (A). Fat deposition was quantified as described under "Materials and Methods" and expressed as a percentage of the total vessel area (B). Data are expressed as means ± S.E. Asterisks (*) represent a p < 0.02 compared with corresponding control mice.

 
Aorta Fat Staining and Cross-section Thickness—The effects of a high fat diet on fat deposition and aorta thickness were measured in L-PGDS KO mice as well as controls. As seen in Fig. 4, the aorta of control C57BL/6 mice had a 2% fat deposition on a low fat diet and a 10% fat deposition after 20 weeks on a high fat diet. This is in sharp contrast to the L-PGDS KO mice, which already had 33 and 50% fat deposition after 20 weeks on the low fat and high fat diets, respectively (Fig. 4).

The effects of the diabetogenic diet on aortic cross-section thickness closely mimicked the fat deposition results. Although the control C57BL/6 aortic thicknesses were unaffected by diet, the L-PGDS KO mice aortic thicknesses were significantly increased after 20 weeks on both the low fat (14% over control) and high fat diets (34% over control) (Fig. 5).

Diabetic Nephropathy and Transforming Growth Factor-{beta} (TGF-{beta}) Staining of Kidney Sections—Microvascular complications of type 2 diabetes such as diabetic nephropathy have been tied to an increase in TGF-{beta} activity (22). Therefore, the effects of a high fat diet on kidney morphology and TGF-{beta} staining were examined. As seen in Fig. 6A, there was a significant increase in C57BL/6 glomerular size as well as increased fibrosis after 20 weeks on the diabetogenic diet. The L-PGDS KO kidneys had significant glomerular hypertrophy and fibrosis even after 20 weeks on the low fat diet, which progressed even further on the high fat diet (Fig. 6A). The C57BL/6 control kidneys had normal tubule formation and almost no TGF-{beta} staining when fed a low fat diet (Fig. 6B). When this same strain was fed a high fat diet we observed the initial stages of tubule damage and fat deposition (red arrows) and TGF-{beta} staining (green arrows). The L-PGDS KO mice already had significantly increased tubule damage and fat deposition as well as higher TGF-{beta} staining on the low fat diet which was exacerbated on the high fat diet (Fig. 6B).

Histopathology results were confirmed by the measurement of serum creatinine after 20 weeks on respective diets. The serum creatinine levels of the L-PGDS KO mice were nearly 2-fold higher than C57BL/6 controls (Fig. 6C).



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  		FIG. 5.
Mouse aorta cross-sections. Control C57BL/6 (n = 9) and L-PGDS KO (n = 9) male mice were sacrificed after 20 weeks on a low fat or high fat diet. Aorta were removed as described under "Materials and Methods" and stained with hematoxylin and eosin (A). Cross-sections were digitized at 20x magnification, and the area of the media was determined and expressed as pixels2 using the SigmaGel program (B). Data are expressed as the means ± S.E. Asterisks (*) represent a p < 0.02 when compared with control mice on a low fat diet.

 
Adiponectin Levels—Adiponectin appears to have substantial anti-inflammatory properties and may be a major modulator of the insulin resistance and increased atherosclerotic risk found in type 2 diabetic patients (23). We measured the level of adiponectin in mice on the low fat and diabetogenic diets and found that there was a significantly lower level of adiponectin in the L-PGDS KO mice compared with control C57BL/6 mice under either dietary condition (Fig. 7).

Adipocyte Size—It is well accepted that muscle and adipose represent the two major tissues of insulin-stimulated glucose disposal. As seen in Fig. 8, visceral adipocytes isolated from C57BL/6 on a high fat diet were 2.5-fold larger in size than those isolated from control mice on a low fat diet. Adipocytes isolated from L-PGDS KO mice on a low fat diet were comparable in size to those isolated from C57BL/6 on a high fat diet and became an additional 40% larger when fed the diabetogenic diet (Fig. 8).

Insulin-stimulated Expression of MKP-2, PTP-1D, and pFAK—Gene array screening of L-PGDS KO and C57BL/6 aorta provided some insight into which signal transduction proteins might be affected by diet (data not shown). We decided to study the effects of insulin stimulation on a select group of these signaling molecules in primary VSMC cultures isolated from the aorta of C57BL/6 and L-PGDS KO mice.

MKP are involved in the feedback regulation of mitogen-activated protein kinase gene expression and have been linked to insulin resistance (24), diabetes (25), and atherosclerosis (26). As seen in Fig. 9A, insulin caused a 2.5-fold stimulation of MKP-2 expression in C57BL/6 (lane 2 versus lane 1), which was further unaffected by exogenously added L-PGDS (lane 3 versus lane 2). The opposite effect of insulin was observed in L-PGDS KO cells. Insulin caused a 40% reduction in MKP-2 expression (lane 5 versus lane 4), which was nearly abolished with exogenously added L-PGDS (Fig. 9A, lane 6 versus lane 5).

FAK phosphorylation, which is involved in growth and migration, has been shown to be affected by insulin (27). We observed a 2-fold increase in insulin-stimulated FAK phosphorylation in C57BL/6 cells (Fig. 9B, lane 2 versus lane 1), whereas there was no change in L-PGDS KO (Fig. 9B, lane 5 versus lane 4). Exogenous L-PGDS stimulated FAK phosphorylation in C57BL/6 cells (Fig. 9B, lane 3 versus lane 2) but actually inhibited FAK phosphorylation in the L-PGDS KO VSMCs (Fig. 9B, lane 6 versus lane 5).



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  		FIG. 6.
Trichrome and TGF-{beta} staining of kidney cross-sections. Control C57BL/6 (n = 8) and L-PGDS KO (n = 8) male mice were sacrificed after 20 weeks on either a low fat or high fat diet. Kidneys were removed and sectioned as described under "Materials and Methods" and either stained with trichrome (A) and viewed at 100x magnification or probed for TGF-{beta} (1 µg/ml) and viewed at 40x magnification (B). Arrows highlight fat deposition (red) and TGF-{beta}(green). C represents the mean serum creatinine ± S.E. after 20 weeks. Asterisk (*) represents a p < 0.01 when compared with controls.

 



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  		FIG. 7.
Adiponectin levels. After 20 weeks on specialized diets, control C57BL/6 (n = 12) and L-PGDS KO (n = 12) male mice were fasted for 5 h. Blood was collected, and serum adiponectin levels were measured as described under "Materials and Methods." Data are expressed as the means ± S.E. Dashed lines represent pre-diet adiponectin levels at 6-8 weeks of age. Asterisks (*) represent a p < 0.05 compared with controls.

 
Insulin signal transduction involves the binding of the insulin receptor to PTP-1D. Expression of PTP-1D was stimulated 3-fold by insulin in C57BL/6 cells (Fig. 9C, lane 2 versus lane 1) yet unaffected in L-PGDS KO cells (Fig. 9C, lane 5 versus lane 4). Exogenously added L-PGDS slightly enhanced PTP-1D expression in C57BL/6 cells (Fig. 9C, lane 3 versus lane 2) and greatly stimulated PTP-1D expression in L-PGDS KO cells (Fig. 9C, lane 6 versus lane 5).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Central obesity is a predisposing factor for the development of type 2 diabetes and is also associated with insulin resistance (28). The precise mechanisms linking a high fat diabetogenic diet and diabetes, however, are unclear. The fact that urinary and serum L-PGDS concentrations increase in people with type 2 diabetes and hypertension led us to examine the role of this protein in diabetes. It is apparent from our data that L-PGDS depletion accelerated glucose intolerance and insulin resistance, since only the L-PGDS KO mice developed these characteristics on the low fat diet (Figs. 1 and 2). That the serum insulin concentrations were elevated in L-PGDS KO mice implies it was the insulin resistance that was responsible for the glucose intolerance rather than any defects in insulin secretion (Fig. 3).

Atherosclerosis and diabetic nephropathy are two common pathologies associated with type 2 diabetes (22, 29). Generally, it is a high cholesterol diet, not a diabetogenic diet, that induces atherosclerosis in the C57BL/6 mouse model (19). It is apparent from our histological data that L-PGDS KO mice had increased fat deposition as well as a thickening of the aortic media, which was not evident in C57BL/6 mice after 20 weeks on the diabetogenic diet (Figs. 4 and 5). This is consistent with our tissue culture observations, where we demonstrated that exogenously added L-PGDS could suppress the hyperproliferation of VSMCs isolated from hypertensive rats and inhibit VSMC migration and cell cycle progression (6, 7). Collectively, these data support the use of thiazolidinediones in the management of diabetes and the associated cardiovascular diseases and may explain why elevated serum levels of L-PGDS predict a decreased occurrence of restenosis (12) and why polymorphisms in L-PGDS result in carotid atherosclerosis (13). In addition, since L-PGDS can bind retinoids, it is quite possible that L-PGDS transports a ligand that activates the retinoid X receptor, which has been reported to reduce atherosclerosis in apoE KO mice (21).

Nephropathy has been associated with the severity of hyperglycemia, which itself may contribute to early alterations in kidney structure and function, before the clinical detection of diabetes (30, 31). Structural changes indicative of diabetic nephropathy, such as glomerular hypertrophy, fibrosis, and basement membrane thickening, were observed in the L-PGDS KO mice on the low fat diet and were further exacerbated on the diabetogenic diet (Fig. 6A). In addition, the deposition of fat and TGF-{beta} was also more pronounced in the L-PGDS KO and appeared in mice fed the low fat diet (Fig. 6B). These data along with the increase in L-PGDS KO serum creatinine (Fig. 6C) would suggest that the L-PGDS KO mouse is a novel model of insulin resistance as well as fat-induced atherosclerosis and diabetic nephropathy.

Adipocyte hypertrophy was obvious in the L-PGDS KOs on either diet (Fig. 8). Although adiponectin levels were significantly lower in C57BL/6 controls (Fig. 7), leptin levels were surprisingly unaffected (data not shown). It should be noted that Fisher et al. (32) have demonstrated that it is the high molecular weight adiponectin complex that better correlates with glucose tolerance. Maier et al. (33) have proposed that soluble N-ethylmaleimide attachment receptor (SNARE) proteins such as syntaxin 4 may be altered in insulin-resistant states and that their levels are modulated by agents that increase insulin sensitivity. Preliminary observations from our laboratory suggest that alterations in the expressions of Glut4 and syntaxin 4 may be involved in the adipocyte hypertrophy observed in L-PGDS KO mice; however, further studies are necessary to confirm these observations. We believe that the role L-PGDS has transporting retinoids is involved in this process since a link exists between the retinoid X receptor and adipogenesis (34).

Reddy et al. (26) have shown that MKP-1 expression is associated with hypercholesterolemia and atherosclerosis and that inhibition of MKP-1 activity may prevent atherosclerotic lesion development in mice. MKP-1 protein is expressed in the atherosclerotic lesions of mice, and inhibition of tyrosine phosphatase activity and MKP-1 protein reduce atherosclerotic lesions in mouse models. Our data demonstrate that basal MKP-2 expression was elevated in L-PGDS KO VSMCs, and the addition of exogenous L-PGDS abolished any MKP-2 expression (Fig. 9A). The fact that exogenously added L-PGDS had no effect on insulin-stimulated MKP-2 expression in C57BL/6 VSMCs is probably related to the fact that the endogenous L-PGDS is at a sufficient concentration to regulate MKP-2.



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  		FIG. 8.
Adipocyte size. Control C57BL/6 (n = 9) and L-PGDS KO (n = 9) male mice were sacrificed after 20 weeks on a low fat or high fat diet. Adipose tissue excised from epididymal fat pads was removed as described under "Materials and Methods" and stained with hematoxylin and eosin (A). Adipocytes were digitized at 40x magnification, and their size was quantitated with the aid of the SigmaGel program. Data are expressed as mean pixels ± S.E. (B). Asterisks (*) represent a p < 0.05.

 
Toriumi et al. (35) have demonstrated that pioglitazone treatment significantly reduced FAK phosphorylation, indicating a novel anti-inflammatory role for this compound. Protein kinase C is an important signaling protein during insulin-mediated cell spreading, and its activation is known to increase FAK-associated phosphatidylinositol 3-kinase activity (36, 37). Previously, we have demonstrated that phorbol ester-induced apoptosis is mediated by L-PGDS phosphorylation and activation by protein kinase C and is accompanied by inhibition of the phosphatidylinositol 3-kinase/protein kinase B anti-apoptotic signaling pathways (38). The observation that insulin-stimulated FAK phosphorylation was enhanced in the presence of L-PGDS leads us to believe that L-PGDS has a definite role in this process, which is absent in the L-PGDS KO strain (Fig. 9B). Furthermore, FAK also regulates insulin-stimulated activity of protein kinase B and glycogen synthase kinase-3{beta} (39), two enzymes we have shown to be regulated by L-PGDS (6).

PTP-1B overexpression decreases glucose uptake as well as GLUT4 translocation to the plasma membrane (40). PTP-1D has also been proposed to be involved in the regulation of glucose uptake and linked to insulin resistance (41). Our data revealed that insulin stimulation led to increased VSMC PTP-1D expression in C57BL/6, which was further enhanced with exogenous L-PGDS (Fig. 9C). The L-PGDS KO VSMCs had low levels of PTP-1D expression which were reversed by exogenous L-PGDS (Fig. 9C). It appears from our signal transduction data that L-PGDS helps sensitize cells to insulin by increasing PTP-1D expression.



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  		FIG. 9.
Insulin-stimulated MKP-2, PTP-1D expressions, and FAK phosphorylation. VSMCs isolated from control male C57BL/6 and L-PGDS KO mice were cultured as described under "Materials and Methods" and serum-starved for 24 h. Cells were pulsed with insulin (10 nM) for 10 min where indicated in the absence or presence of a 2 h L-PGDS (50 µg/ml) pre-incubation. The proteins were extracted as described under "Materials and Methods," and the blots were probed with antibody to MKP-2 (A), phosphor-FAK/FAK (B), or PTP-1D (C). Representative blots from at least three experiments are provided.

 
In this study we have demonstrated an important role for L-PGDS in diet-induced diabetes using mice deficient in L-PGDS expression. We propose that the elevation of L-PGDS in diabetes and hypertension is a compensatory mechanism to correct insulin resistance and alleviate the cardiovascular problems associated with the diseases by suppressing vessel media overgrowth. Furthermore, we propose that the L-PGDS KO mouse represents a model of insulin resistance and diabetic nephropathy as well as fat-induced atherosclerosis.


   FOOTNOTES
 
* This work was supported by an American Diabetes Association Career Development Award, National Institute of Health Grant R01HL06 7953-01A2, and the Winthrop-University Hospital Department of Medicine. 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. Back

** Present address: Waseda-Olympus Bioscience Research Institute, Helios, 05-01/02, 11 Biopolis Way, Singapore 138667. Back

To whom correspondence should be addressed: Vascular Biology Institute, Winthrop-University Hospital, 222 Station Plaza North, Suite 505-B, Mineola, NY 11501. Tel.: 516-663-2028; Fax: 516-663-4710; E-mail: lragolia{at}winthrop.org.

1 The abbreviations used are: VSMC, vascular smooth muscle cell; L-PGDS, lipocalin-type prostaglandin D2 synthase; PG, prostaglandin; KO, knock-out; PTP-1D, phosphotyrosine phosphatase-1D; MKP, mitogen-activated protein kinase phosphatase; FAK, focal adhesion kinase; TGF-{beta}, transforming growth factor-{beta}. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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