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Published as doi: 10.1096/fj.05-4617fje.
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(The FASEB Journal. 2006;20:1203-1205.)
© 2006 FASEB

Adipose-specific effect of rosiglitazone on vascular permeability and protein kinase C activation: novel mechanism for PPAR{gamma} agonist’s effects on edema and weight gain

Konstantinos B. Sotiropoulos, Allen Clermont, Yutaka Yasuda, Christian Rask-Madsen, Motonobu Mastumoto, Junichi Takahashi, Kim Della Vecchia, Tatsuya Kondo, Lloyd P. Aiello and George L. King1

Research Division, Joslin Diabetes Center, Harvard Medical School One Joslin Place, Boston, Massachusetts, USA

1Correspondence: Joslin Diabetes Center, One Joslin Place, Boston, MA 02215, USA. E-mail: george.king{at}joslin.harvard.edu

ABSTRACT

PPAR{gamma} agonists, thiazolidinediones, cause fluid retention and edema due to unknown mechanisms. We characterized the effect of rosiglitazone (RSG), a thiazolidinedione, to induce vascular permeability, vascular endothelial growth factor (VEGF) expression, and protein kinase C (PKC) activation with edema and wt gain. In lean, fatty and diabetic Zucker rats, and endothelial insulin receptor knockout mice, RSG increased wt and vascular permeability, selectively in fat and retina, but not in heart or skeletal muscle. H2O content and wt of epididymal fat were increased by RSG and correlated to increases in capillary permeability in fat and body wt. RSG induced VEGF mRNA expression and PKC activation in fat and retina up to 2.5-fold. Ruboxistaurin, a PKCß isoform inhibitor, in the latter 2 wk of a 4-wk study, normalized vascular permeability in fat and decreased total wt gain, H2O content, and wt of fat vs. RSG alone but did not decrease VEGF expression, basal permeability, or food intake. Finally, RSG did not increase wt or vascular permeability in PKCß knockout vs. control mice. Thus, thiazolidinedione’s effects on edema and wt are partially due to an adipose tissue-selective activation of PKC and vascular permeability that may be prevented by PKCß inhibition.—Sotiropoulos, K. B., Clermont, A., Yasuda, Y., Rask-Madsen, C., Mastumoto, M., Takahashi, J., Della Vecchia, K., Kondo, T., Aiello, L. P., King, G. L. Adipose-specific effect of rosiglitazone on vascular permeability and protein kinase C activation: Novel mechanism for PPAR{gamma} agonist’s effects on edema and weight gain.


Key Words: Zucker diabetic fatty rat • ruboxistaurin • vascular endothelial growth factor • insulin receptor knockout mice • thiazolidinedione

THIAZOLIDINEDIONES (TZDS) INCREASE insulin sensitivity (1) by binding and activating the transcription factor peroxisome proliferator-activated receptor (PPAR) {alpha} and {gamma}. TZDs are thought to enhance insulin’s effects through PPAR{gamma}· (2) via activation of PI3 kinase pathways to mediate insulin’s metabolic actions in many tissues. PPAR{gamma}· is expressed at high levels in adipose tissues, colon, and activated macrophages, and at lower levels in vascular and other tissues (3 , 4) . PPAR{gamma} and its agonist ligands can stimulate adipose differentiation (5) , leading to increased numbers of small adipocytes, which are believed to be more insulin-sensitive than large, lipid-laden adipocytes (6) . Improvement of insulin’s actions directly on the liver and skeletal muscle have also been reported (7 8 9) .

Serious adverse effects of TZDs, such as wt gain and edema, have limited its clinical use, particularly in diabetic patients with symptoms of angina and congested heart failure (10) . In clinical trials with rosiglitazone (RSG) and pioglitazone, the frequency of edema in TZD-treated patients was ~3–4 times higher than in placebo-treated patients. When used in combination with insulin, the prevalence of edema can be as high as 16% (11) . The increase in body wt may reflect both fat accumulation and fluid retention in patients taking TZDs. For wt gain, the cause is likely multifactorial, resulting from factors such as improved glycemic control, differentiation of adipocytes, increased body fat, increased appetite, and fluid retention (11 , 12) .

The pathogenesis of TZD-induced edema could involve renal tubules or peripheral tissues. Within the 6 to 12 wk of treatment, a modest wt gain and small reductions in hemoglobin and hematocrit occur in most patients (12 , 13) . Increases in renal sodium retention and vascular permeability induced by TZDs have been suggested (14) . However, PPAR{gamma} agonist GI262570 does not appear to exert any direct effect on renal hemodynamics (15) . TZD-induced edema could also be a result of increased vascular permeability. RSG has been reported to directly increase permeability across a layer of pulmonary endothelial cells (16) . In addition, TZDs have been shown to increase the expression of VEGF (17) , which is a powerful permeability-inducing agent. In this study, we have characterized the effect of RSG on vascular permeability in the retina, heart, skeletal muscle, and fat from animal models of insulin resistance and diabetes. In addition, we have explored the potential involvement of two well-established vascular permeability factors—vascular endothelial growth factor and protein kinase C (PKC) activation—on TZD-induced edema. PKC activation has been reported to be increased in vascular tissues of diabetic and insulin-resistant animals and can alter endothelial barrier functions by phosphorylating cytoskeletal proteins forming the intercellular tight junctions (18 19 20) . Conversely, inhibitors of PKC, especially the ß isoform, have been demonstrated to reduce retinal vascular permeability induced by diabetes or VEGF (19) . Lastly, we also determined whether insulin’s effects on the endothelial cells have a role on regulating capillary permeability since insulin treatment in combination with TZDs significantly increases the risk of edema (21) .

MATERIALS AND METHODS

Animals
Rats
Male Zucker lean (fa +/+) (ZL, n=58), insulin-resistant obese fa/fa rats (ZF, n=73, Harlan, Indianapolis, IN) and diabetic fatty rats (ZDF, n=19, Genetic Models Inc, Indianapolis, IN) were used at 14–16 wk of age. Physiological parameters of these rats are presented in Table 1 .


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  		Table 1. Effects of rosiglitazone (RSG) treatment on the general characteristics and biochemical parameters of Zucker rats

Mice
Vascular endothelium–specific insulin receptor knockout (VENIRKO) mice, which were generated using the Cre-loxP system, were provided by Dr. C. Ronald Kahn as described previously (22) . Due to complex breeding, mice have a mixed genetic background of 129SV, C57B/6, SJL, FVB, and DBA strains. To minimize the difference in the genetic background, littermates from the same breeding pairs were used at the age of 10–12 wk.

PKCß knockout (KO) mice were produced as described previously and provided by Dr. Michael Leitges (23) . PKCß KO mice have a mixed background of 129SV and C57/B6 strains. Age-matched control mice, F2 hybrid, B6/129S were purchased from the Jackson Laboratory, Bar Harbor, ME. PKCß KO and their control mice were used at 10–12 wk of age.

Drug treatments
All animals were housed on a 12-h light/dark cycle and fed standard powdered rodent chow (LabDiet, Richmond, IN). Treated groups had their chow supplemented with rosiglitazone maleate (RSG) (GSK, London, UK) at the dose of 50 µM/ kg (0.00236% in chow diet) for 4 wk. In the PKCß inhibitor treatment group, animals received ruboxistaurin (RBX or LY333531) (Eli Lilly, Indianapolis, IN) in their chow at the dose of 0.053% in diet for the last 2 wk of the 4-wk study (24) or for 2 wk alone to study the effects of RBX on basal wt and capillary permeability. All protocols for animal use and euthanasia were approved by the Animal Care Committee of Joslin Diabetes Center and were in accordance with NIH guidelines.

In Zucker rats, body wt and blood glucose (Glc) levels were determined at baseline, after 2 wk of treatment, and at the end of the study after 4 wk of treatment, while food intake was being monitored throughout the study. Blood pressure was measured at baseline and at the end of the study. Biochemical parameters were measured at the end of the study when vascular permeability assessed by EB dye method. At the end of the study, monocytes were isolated from blood drawn from the rats and tissues were obtained for measurements of water content and VEGF expression. Finally, retinal blood flow and mean circulation time were measured in Zucker lean rats treated with RSG for 4 wk and controls.

In VENIRKO mice and PKCß KO mice, wt, food consumption, and EB vascular permeability were assessed in a similar manner. Furthermore, in VENIRKO and control mice, CLAMS assessments were performed after 4 wk of treatment (regular food, RSG alone or RSG+RBX).

Blood pressure measurement
Blood pressure was measured by tail cuff photoelectric plethysmography in restrained, unanesthetized rats using a UR-5000 UEDA system (UEDA Electronic Works Ltd., Tokyo, Japan). Systolic (SBP), mean (MBP), and diastolic blood pressure (DBP), and heart rate (HR) were obtained as the average of 20–30 measurements that showed stable readings over 10–15 min.

Measurements of blood Glc, hematocrit, insulin, and lipids in rats
Blood Glc was measured by Glucometer Elite (Bayer, Germany). Hematocrit, plasma insulin, triglycerides, total cholesterol, HDL, and LDL cholesterol were measured by clinical chemistry laboratory at Joslin Diabetes Center, Boston, MA.

Measurement of vascular permeability with Evans blue dye
Vascular permeability was quantified using Evans blue (EB) dye. This dye noncovalently binds to plasma albumin and is a marker for proteins around 67 kDa (25) . Under deep anesthesia, EB dye blue was injected from a heparinized PE50 catheter inserted in the right jugular vein. After additional circulation for 120 min in the rats or 60 min in the mice, each animal was perfused with citrate-buffered 1% paraformaldehyde (37°C) and normal saline for 2 min to clear the dye from left ventricle with the physiological pressure kept at 100 mmHg. Eyes were enucleated immediately and the retinas were carefully dissected under an operating microscope. Heart, epididymal fat pads, perirenal fat, and soleus muscle were dissected and 10–20 mg of tissues was put into preweighed tubes. Remaining tissues were flash frozen with liquid N2 and kept at –80°C. In some experiments, whole epididymal fat pads were removed, weighed, and used for the isolation of microvessels as described previously (26) .

The wt of each tissue was measured wet and dry after being desiccated in Speed-Vac (Savant Inc., Holbrook, NY) for up to 8 h. Albumin leakage into the tissues was estimated via the measurement of extravasated EB dye, which was extracted by incubating each tissue in 0.2 ml formamide for 18 h at 70°C. Extracts were filtered through a MW 30 kDa filter in a centrifuge at 3000 g for 60 min. at 4°C. EB dye was quantified by measuring the absorbance of the filtrates at 620 nM for maximum and 720 nM for reference. Blood samples were centrifuged at 10 000 g for 20 min at 4°C, and the concentration of the EB dye in the supernatants was calculated from a standard curve of EB dye in formamide. The concentration of EB dye in the extracts was calculated from a standard curve and normalized with the dry wt and the concentration of EB in the plasma.

Water content of tissues
Wet and dry weights of the tissue samples processed for EB leakage measurements were used to calculate the water content of the tissue by the following formula: Water content = (wet wt – dry wt)/ wet wt x 100.

Mononuclear cells isolation
Blood was drawn under anesthesia from vena cava and was kept in tubes containing sodium citrate as an anticoagulant. Isolation of mononuclear cells was done by density gradient. Cells were washed three times with PBS and used for PKC activity measurements.

Real-time quantitative RT-PCR for VEGF
Total RNA was extracted from frozen tissues and isolated by TRI-REAGENT (Molecular Research Center Inc., Cincinnati, OH). For adipose tissue samples, the surface oil layer was removed prior to chloroform extraction to ensure RNA quality. Primers and probes for VEGF were designed using Primer Express 1.5a software (ABI Prism; Perkin-Elmer Applied Biosystems, Foster City, CA) with the following sequences: 5' primer, 5'-CGC AAG AAA TCC CGG TTT AA -3'; 3' primer, 5'-CAA ATG CTT TCT CCG CTC TGA-3'; and probe, 6FAM-TCC TGG AGC GTT CAC TGT GAG CCT T-TAMRA. Real-time quantitative RT-PCR was performed with the TaqMan One-Step RT-PCR Master Mix Reagents kit (Perkin-Elmer Applied Biosystems) and the ABI Prism 7700 sequence detection system (Perkin-Elmer Applied Biosystems) with the following cycle profile: 1 cycle at 48°C for 30 min, 1 cycle at 95°C for 10 min, 40 cycles at 95°C for 15 s, and 60°C for 1 min. As an internal control, rodent GAPDH gene and 18S RNA (rodent GADPH or rRNA control reagents, Perkin-Elmer Applied Biosystems) were used for normalization. For comparison, the expression concentration of these genes in the ZL rats or the control mice was arbitrarily set at 1.

Measurement of in situ PKC activity in mononuclear cells and rat retina
The measurement of PKC activity in situ of mononuclear cells (~2–4x106 cells), and the whole retina was performed using the method described previously (24) .

Measurement of PKC activity in membranous and cytosolic fractions of microvessels isolated from the fat
PKC activity in the membranous and cytosolic fractions of the microvessels was measured by assessing the transfer of 32P from [{gamma}-32P]-ATP (3000 Ci/mmol; New England Nuclear, Boston, MA) into specific substrate octapeptide (RKRTLRRL), in the presence of Ca2+, phosphatidylserine (PS), diacylglycerol (DAG) (Avanti Polar Lipids, AL) as described previously (27) .

Immunoblot analysis
Samples were processed for immunoblot analysis as described before (24) . Membranes were immunoblotted with anti-CD34, anti–PECAM-1 and antiactin antibodies (Santa Cruz Biotechnology, San Diego, CA).

Assay of total DAG
Total DAG levels were measured with a radioenzymatic assay kit (Amersham Corp., Arlington Heights, IL) using DAG kinase that quantitatively converts DAG to [32P] phosphatidic acid (PA) in the presence of [{gamma}-32P]-ATP (New England Nuclear, Boston, MA). The values of total DAG contents were normalized by the amount of cellular proteins.

Comprehensive lab animal monitoring system (CLAMS)
Control and VENIRKO mice being treated for 4 wk were allowed to acclimate for 1 d in a polycarbonate metabolic cage before being monitored. CLAMS were performed in the Animal Physiology Core of Joslin Diabetes Center (Columbus Instruments, Columbus, OH). Results were analyzed by manufacturer software Oxymax.

Mean circulation time (MCT) and retinal blood flow (RBF)
Video fluorescein angiography was used to determine RBF. The video fluorescein angiography system has been previously described (28) .

Statistical analyses
Results were expressed as mean ± SE (SE). Comparisons were performed by t-test or one-way ANOVA with Student-Newman-Keuls methods. P-values of <0.05 were defined as statistically significant. Paired t test was used for analysis of total wt gained at 2-wk intervals before and after the initiation of treatments with either RSG or RBX.

RESULTS

Effect of rosiglitazone (RSG) on physiology of ZL, ZF, and ZDF rats
Body wt of untreated ZL, ZF, and ZDF rats progressively increased and attained final weights of 401 ± 5, 603 ± 7, and 432 ± 22 g, respectively. Treatment with RSG significantly increased wt gain in all rat groups compared with those fed with the regular chow (Table 1) . Untreated ZL and ZF rats had significantly higher systolic BP by the end of the study vs. their baseline levels (basal BP (in mmHg): ZL 138±2 and ZF 143±3, P =0.04 and P =0.03, respectively). RSG treatment prevented the increases in systolic BP in ZL, ZF, and ZDF rats (Table 1) . Diastolic BP of ZF and ZDF rats was increased when compared with ZL. RSG treatment decreased diastolic BP in both ZF and ZDF rats (P =0.04 and P =0.02 vs. ZF and ZDF controls, respectively) (Table 1) . No changes were observed in the heart rate after RSG treatment. Untreated ZL and ZF had higher blood Glc levels at the end of the 4-wk study compared with those at baseline [baseline blood Glc (mg/dl): ZL 92±1 and ZF 113±3, P=0.04 and P =0.02, respectively]. RSG lowered blood Glc in ZL, ZF, and ZDF rats, but the decreases in ZDF did not reach significance (Table 1) . Plasma insulin levels were significantly higher in ZF rats than in ZL and ZDF rats. RSG decreased insulin levels in ZF-treated rats vs. untreated controls (7.8±1.1 vs. 27.9±5.3 ng/ml) (P =0.003) while no change was observed in RSG-treated ZL or ZDF rats (Table 1) .

Plasma triglycerides levels were reduced significantly in all groups treated with RSG. (Table 1) . Hematocrit was not altered by RSG treatment in ZL and ZF rats but a significant decrease was observed in ZDF rats after RSG treatment (P =0.05, vs. untreated ZDF, Table 1 ).

Vascular permeability to Evans blue (EB) dye
Permeability of EB dye across the endothelial barrier was examined in epididymal and perirenal fat, retina, heart and skeletal muscle (Fig. 1 ). In epididymal fat, EB extravasation per dry wt of tissue was higher in the untreated ZL vs. ZF and ZDF (by 45 and 35% respectively, P <0.001) (Fig. 1A ). At basal, ZDF had a greater leakage than ZF (P =0.03). RSG treatment significantly increased vascular permeability in the epididymal fat by 30% (P <0.001), 50% (P <0.001), and 20% (P =0.04) in ZL, ZF, and ZDF, respectively, when compared with their untreated controls. Similar changes caused by RSG were observed in perirenal fat (data not shown). For the retina, EB leakage was again lower in untreated ZF rats than in ZL and ZDF rats (P <0.001 and P =0.007, respectively). When treated with RSG, the amount of EB dye leakage was significantly increased in the retina of ZL (by 30%) and ZF rats (by 40%) (P =0.04 and P =0.02 vs. their control group), but not in ZDF rats (Fig. 1B ).


Figure 1
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  		Figure 1. Effect of rosiglitazone on vascular permeability of EB dye in (A) epididymal fat, (B) retina, (C) heart, and (D) soleus muscle from Zucker lean (LEAN), fatty (FATTY) or diabetic fatty (ZDF) rats treated with control (CTL) chow or chow containing rosiglitazone (RSG); n = Number of rats studied. Results are plotted as mean ± SE. Detailed descriptions are provided in the Methods section. For comparison, the amount of EB dye in the tissue of the LEAN rats fed with CTL chow was arbitrarily set to 100%.

Extravasation of EB in the heart of untreated ZL and ZF rats was significantly higher compared with ZDF controls (P =0.001 and P =0.03, respectively), but surprisingly RSG treatment did not alter EB dye permeability in the myocardium (Fig. 1C ). Similar to the myocardium, EB dye permeability in the soleus muscle was higher in ZL than in ZF (P =0.04) and ZDF (P =0.02) at basal, but again RSG did not induce any changes (Fig. 1D ).

Water content
The water contents of the different tissues were also measured to determine whether increases in vascular permeability have any biological consequences. Relative to other tissues, total water content/wet wt in the adipose tissue (10%) was much lower than in the retina, myocardium and skeletal muscle ({approx}80%), as expected (29) . In the epididymal fat, untreated ZL had higher water content/wet wt than ZF (P =0.04). Percent of water content was increased by RSG treatment in the epididymal fat by 30, 30, and 10% in ZL, ZF, and ZDF rats, respectively, vs. their controls (P =0.04, P=0.007 and P =0.04, respectively) (Fig. 2 A).


Figure 2
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  		Figure 2. Percent of water content in the (A) epididymal fat and (B) retina from Zucker lean (LEAN), fatty (FATTY) or diabetic fatty (ZDF) rats treated with control (CTL) chow or chow containing rosiglitazone (RSG). Results are plotted as mean ± SE; n = number of rats studied. Description of the procedures and the calculation of percent of water content are stated in Materials and Methods.

In the retina, water content did not differ among the three untreated rat groups. RSG increased water content in the retina of ZF rats as compared with the control (P =0.05), whereas no changes were induced by RSG in ZL and ZDF groups (Fig. 2B ). Similar to EB permeability, RSG treatment did not cause any significant changes in water content/wet wt of the heart and the soleus muscle, although water content/wet wt in soleus muscle was lower in both ZF and ZDF (P =0.02 for both) rats as compared with ZL rats (data not shown).

VEGF expression
Basal VEGF mRNA expression was 50% lower in the epididymal fat of both ZF and ZDF rats as compared to ZL rats (P =0.02 and P =0.002 respectively) (Fig. 3 A). RSG treatment increased VEGF mRNA expression by 1.4, 2.2, and 2 fold for ZL, ZF, and ZDF, respectively (P =0.02, P =0.01, and P =0.04, respectively). Unlike adipose tissues, ZDF rats had significantly higher retinal VEGF mRNA expression than ZL rats (P <0.001), consistent with previous reports (30) (Fig. 3B ). RSG significantly increased VEGF expression in the retina of ZL and ZF rats (P =0.05) but the increase in VEGF expression in ZDF rats did not reach significance. In contrast to the retina, VEGF expression in the myocardium in untreated ZF and ZDF rats was lower than in ZL rats (P =0.04 and P =0.03, respectively), as previously reported (30) (Fig. 3C ). RSG did not significantly change VEGF expression in the myocardium or soleus muscle. At basal, ZDF rats had significantly lower VEGF mRNA expression in the soleus muscle as compared to ZL and ZF (0.02 and 0.002, respectively) (Fig. 3D ).


Figure 3
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  		Figure 3. Expression of VEGF mRNA in (A) epididymal fat, (B) retina, (C) heart, and (D) soleus muscle in Zucker lean (LEAN), fatty (FATTY), and diabetic fatty (ZDF) rats treated with control (CTL) chow or rosiglitazone (RSG) chow. VEGF mRNA levels were quantified by real-time RT-PCR as described in Materials and Methods and plotted as mean ± SE; normalized to VEGF mRNA levels of the LEAN rats fed with CTL chow; n = number of rats studied. P-value calculated as described in the Statistics section of Materials and Methods.

Effect of RSG on capillary density and retinal blood flow
One potential explanation for TZD-induced extravasation of EB dye in adipose tissue could be increases in capillary density. RSG treatment’s effect on the density of capillaries in the epididymal fat, as estimated by measuring the protein expression of endothelial markers, CD34 and PECAM-1 (31) , did not differ in the RSG-treated and untreated rats. However, protein expression of CD34 and PECAM-1 were 20–30% lower in ZF rats compared with ZL and ZDF rats. Further, retinal blood flow was measured in ZL rats with and without RSG treatment by video fluoroscein angiogram. No changes were induced by RSG treatment.

DAG-PKC activity of microvessels isolated from fat
The levels of DAG and PKC activities were measured in the microvessels of epididymal fat and the retina since DAG/PKC can regulate vascular barrier and are known to be activated in insulin resistant and diabetic states (24 , 32 33 34) . DAG levels in untreated ZF rats were significantly elevated compared to ZL rats by >2 fold (P =0.01). RSG treatment increased DAG levels further in both ZL (65%, P =0.04) and ZF (70%, P=0.05) (Fig. 4 A).


Figure 4
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  		Figure 4. Effect of rosiglitazone on (A) DAG levels and (B) PKC activities from microvessels of adipose tissue, (C) retina, and (D) mononuclear cells in Zucker lean (LEAN) and fatty (FATTY) rats treated with control (CTL) chow or rosiglitazone (RSG) chow. Procedures for tissue isolation and assays for DAG/PKC activities are described in Materials and Methods; n = Number of rats studied.

PKC activities of the membrane and cytosol fractions of epididymal microvessels of ZL and ZF rats with and without RSG treatment were measured. RSG treatment increased PKC activities in the membrane fractions (activated pool) of ZL (by 45%) and ZF (by 35%) rats (P =0.08 and P =0.07, respectively) (Fig. 4B ), while no changes of PKC activities were observed in the cytosol fraction (data not shown). For the retina, RSG treatment increased in membranous PKC activities in ZL and ZF rats vs. their controls (P =0.06 and P <0.05, respectively) (Fig. 4C ).

In contrast, RSG treatment decreased the activated pool of PKC activity in the mononuclear cells isolated from ZL and ZF rats (by 25 and 40%, respectively, P =0.03 for ZF) as shown in Fig. 4D . Interestingly, the activated PKC fractions were increased in the untreated ZF rats by 80% as compared with ZL rats (P =0.01) in the mononuclear cells (Fig. 4D ).

Effect of PKCß isoform inhibitor, RBX, on RSG-induced changes
Since RSG treatment increased DAG and, likely, the activated pool of PKC in the epididymal microvessels, we studied the effect of PKCß selective inhibitor, ruboxistaurin (RBX) to inhibit RSG’s effects on vascular functions (24) . Similar to the results above, RSG for 4 wk increased the permeability of EB dye in the epididymal fat of ZL and ZF rats by 40 (P =0.01) and 60% (P =0.01), respectively (Fig. 5 A). The addition of RBX in the last 2 wk of the 4-wk RSG treatment significantly decreased EB dye leakage compared with RSG alone in the epididymal fat (P =0.02 and P =0.03 vs. RSG-treated ZL and ZF, respectively). Similar trends were observed in the retina where combined treatment with RBX decreased EB dye leakage by 20 and 25% compared with RSG-treated ZL and ZF rats, although these changes were not significant (data not shown). RBX treatment alone for two weeks did not affect EB dye permeability in fat tissues from the epididymus, subcutaneous (s.c.) fat, or the retina at basal state in ZL rats.


Figure 5
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  		Figure 5. Effect of rosiglitazone (RSG) and ruboxistaurin (RBX) on (A) EB dye permeability and (B) total water content in epididymal fats. Zucker lean (LEAN) and fatty (FATTY) rats were treated with regular chow (CTL) and RSG (RSG) for 4 wk and then studied. A separate group of rats was treated with RSG for 2 wk and another 2 wks with chow containing RSG and RBX; n = number of rats studied. Values plotted are mean ± SEM with P-values calculated as described in Materials and Methods.

Total water contents in the epididymal fat of ZL and ZF rats were increased after RSG treatment vs. untreated ZL and ZF rats. The addition of RBX normalized the total water content of the epididymal tissues in RSG-treated ZL and ZF rats with the change in ZF rats induced by RBX reaching significance (P =0.03, Fig. 5B ). No significant changes were induced by RBX in the water content of retina, heart, or soleus muscle in ZL or ZF rats (data not shown).

ZL and ZF rats treated with RSG alone for 4 wk had increased wt gains of 38 ± 3 g and 86 ± 7 g, respectively, vs. untreated ZL (P =0.001) and ZF (P <0.001) (Fig. 6 A). However, when compared with groups treated with RSG alone, rats treated with RBX+RSG for the last 2 wk had significantly lower weights for ZL (P =0.05) and ZF (P=0.03), respectively (Fig. 6A ). In fact, for the first 2 wk the RSG-treated rats had similar wt gains of 24 ± 2 g (RSG) and 23 ± 2 g (RSG+RBX) in ZL and 53 ± 5 g (RSG) and 53 ± 4 g (RSG+RBX) in ZF since RBX was not added until the latter 2 wk. The addition of RBX for the last 2 wk of the study significantly reduced the wt gain of both ZL (P =0.01) and ZF rats (P =0.002) compared with the RSG-alone treated groups (Fig. 6B, C ). Besides total wt, RSG treatment increased the weights of epididymal fat pads for both ZL (40%) and ZF (65%) significantly (P<0.001) vs. their untreated controls. The addition of RBX to RSG treatment reduced the wt of fat pad when compared to RSG alone for ZL (P =0.05) and ZF rats (P =0.04) (Fig. 6D ). To exclude the possibility that RBX’s effect was due to changes in food consumption, food intake of the rats was monitored. RSG treatment alone significantly increased food intake in ZL and ZF rats by 10 and 25%, respectively. RBX treatment did not alter food intake or wt of ZL or ZF rats with or without treatment with RSG (data not shown).


Figure 6
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  		Figure 6. wt gains in response to rosiglitazone (RSG) or RSG+ruboxistaurin (RBX) treatment. A) Total wt gains and (D) total wt of epididymal fat pads in Zucker lean (LEAN) and fatty (FATTY) rats. Weight gains in the first and second 2 wk of the study in (B) LEAN and (C) FATTY rats; n = number of rats studied. CTL = rats treated with regular rat chow. RSG = rats fed with RSG for 4 wk. RSG+RBX = rats treated with RSG for 2 wk and then RSG+RBX for another 2 wk.

Effect of PKCß inhibitor, RBX, on VEGF expression
RSG treatment alone for 4 wk increased VEGF mRNA expression in the epididymal fat, measured using quantitative RT-PCR, by 70% in ZL (P =0.01) and by 260% in ZF rats (P=0.001). However, combined treatments with RBX and RSG for the last 2 wk of the study did not decrease VEGF mRNA expression significantly in either ZL or ZF rats (Fig. 7 A). Similar findings were obtained in the retina where RSG treatment alone increased VEGF mRNA levels significantly in both ZL and ZF rats, which were not decreased by treatments with RSG+RBX (Fig. 7B ).


Figure 7
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  		Figure 7. VEGF mRNA expression in Zucker lean (LEAN) or fatty (FATTY) rats treated with control (CTL) chow, rosiglitazone (RSG) alone or RSG treated for 2 wk, and RSG+ruboxistaurin (RBX) treated for another 2 wk. Results plotted are mean ± SEM in (A) epididymal fat or (B) the retina; n = number of rats studied. Procedures for isolation of the tissues and quantitation of VEGF mRNA levels by real-time RT-PCR are described in Materials and Methods.

Effect of RSG treatment on PKCß knockout (KO) mice
To confirm the role of PKCß isoform in RSG-induced increases in vascular permeability, the effect of RSG was studied in PKCß KO mice (23) . Permeability of EB dye was similar in wild-type (WT) and PKCß KO mice in the epididymal fat and the retina at basal state. RSG treatment for 4 wk increased the EB leakage in adipose tissue by 70% (P =0.04) and the retina by 50% in the control mice, but not in the PKCß KO mice (Fig. 8A, B ). wt gain in WT mice treated with RSG was significantly greater than in untreated WT mice (P =0.02). RSG treatment did not induce wt gain of PKCß KO mice (Fig. 8 C).


Figure 8
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  		Figure 8. Effect of treatment of control (CTL) or rosiglitazone (RSG) diet on control or PKCß KO mice (both C57/B6/129S background) for 4 wk on vascular permeability to EB dye in (A) epididymal fat and (B) the retina, or (C) total body wt; n = number of mice studied. Results are plotted as mean ± SEM

Role of endothelial cell insulin receptor in vascular permeability
The role of insulin receptors on the vascular permeability of endothelial cells was studied because the combined use of insulin and TZD resulted in a higher prevalence of edema than TZD alone (11) . The effects of RSG on EB dye permeability were examined in vascular endothelium specific insulin receptor knock out mice (VENIRKO) (22) . Permeability of EB dye did not differ between VENIRKO and WT control mice at basal state (Table 2 ). RSG treatment increased EB dye permeability in the epididymal (Table 2) and s.c. fat (not shown) of both VENIRKO by 35% (P =0.03) and control mice by 45% (P =0.03). RBX treatment in RSG-treated VENIRKO and control mice for the last 2 wk of the 4-wk study, decreased EB permeability compared to RSG alone (P =0.01 and P =0.04 in epididymal, for control and VENIRKO, respectively). Similarly, RSG treatment increased EB leakage in the retina equally in control and VENIRKO mice, which was normalized by the addition of RBX for the last 2 wk of the study (data not shown).


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  		Table 2. Effects of rosiglitazone (RSG) and ruboxistaurin (RBX) on vascular permeability to Evans Blue leakage and water content of epididymal fat pads, and weight gain, in wild-type and VENIRKO mice

Similar to the Zucker rats, RSG treatment increased water content in the adipose tissues in both WT and VENIRKO mice (P =0.03 and 0.05, respectively), which was reduced by RBX treatment (Table 2) . Weight gains of VENIRKO and WT mice treated with RSG were increased significantly compared to untreated groups (P =0.01 and P =0.05, respectively) and higher than the combined RBX and RSG-treated group (P =0.05 in WT mice, Table 2 ). Food consumption was similar in RSG and RSG+RBX groups but was higher compared with untreated groups. No changes in physical activities of WT and VENIRKO mice with or without RSG treatment were detected by CLAMS. Hence, endothelial insulin receptor is not required for the effects of RSG on vascular permeability.

DISCUSSION

In the current study, RSG induced many metabolic effects in ZF and ZDF rats, which were expected in accordance with its insulin-sensitizing effects, such as decreases in plasma levels of Glc, insulin, and triglycerides, and increases of HDL-cholesterol and body wt (35) . RSG also had expected hemodynamic effects such as lowering systolic and diastolic BP and decreasing hematocrit due to hemodilution (10) . Thus, the physiological response of ZF and ZDF rats to RSG suggests this rat model approximated some of TZDs’ effects in type 2 diabetic patients both in metabolic and hemodynamic changes.

The results derived from the use of EB dye, which binds to albumin and thus measures capillary permeability of albumin, suggested that RSG could increase vascular permeability in selected tissues (28) . Interestingly, this effect of RSG was not general, but appeared to be tissue selective since it was observed mainly in the adipose tissues and retina, but not in the myocardium, skeletal muscle, and liver (data not shown).

The tissue selectivity of RSG’s effect on vascular permeability has not been reported previously. However, the results that EB extravasation in the retina and adipose tissues were increased in the diabetic state (ZF vs. ZDF) are consistent with reports of increased vascular permeability in diabetic patients and rodent models of diabetes (36) . The decreases in permeability observed in the adipose tissues from insulin-resistant states are new findings, which are likely due to the hypertrophy of the adipose cells and fat mass, leading to decrease in capillary density. However, the retina also exhibited decreases in capillary permeability in insulin-resistant states without exhibiting changes in capillary density. It is not known whether fat content could also be increased in the retina, as in adipose tissues. It is also possible that insulin resistance could have changed retinal hemodynamic to decrease permeability, although systolic BP was actually increased and measurements of retinal blood flow were unchanged in ZF rats compared to ZL rats, as reported by us previously (28) . Further studies are needed to clarify the mechanism and biological significance of the decrease in basal capillary permeability in the retina and adipose tissues in insulin-resistant states.

The tissue-specific effect of RSG on vascular permeability was confirmed by parallel increases in water content and weights of epididymal fat pads and total body wt. Adipose tissues exhibited total water content at 10% of its wt as expected due to large amount of endogenous lipid, compared with other tissues that exhibited total water content at {approx}80% (29) . RSG increased water contents in ZL, ZF, and ZDF, suggesting that insulin sensitivity of the tissue may not be an essential factor in RSG-induced edema. This conclusion is supported by the results in the VENIRKO mice, which responded to RSG with increases in permeability and wt gain comparable to its controls, indicating that insulin effects on endothelial cells and insulin resistance are not essential for TZD to induce edema.

One possible mechanism for TZD’s effect on fluid retention is related to increases in the expression or actions of VEGF, which can increase vasodilation and permeability (37) . Our results support the idea that TZDs can increase VEGF expression in many tissues including vascular and nonvascular origin. Previous reports have indicated that both troglitazone and pioglitazone can increase VEGF expression in vascular smooth muscle cells (17) . However, this effect of TZD on VEGF expression is not uniformly observed since Panigraphy et al. reported that rosiglitazone reduced VEGF expression in tumor cells (38) . It is not clear from our results whether the increases in VEGF expression play a significant role in TZD’s induction of edema and capillary permeability. In addition, RBX treatment decreased permeability, edema and wt gain, but did not reduce VEGF expression in the adipose tissue (Fig. 7A ).

Thus, TZDs may also stimulate edema due to VEGF independent pathways. Interestingly, diabetes also did not uniformly increase the expression of VEGF in all tissues. Our results confirmed that diabetes increased VEGF expression in the retina, but paradoxically decreased VEGF mRNA expression in the heart and skeletal muscle (30) . In the retina, RSG increased both capillary permeability and VEGF expression mainly in the ZF rats and not in the ZDF rats. This may be due to the fact that VEGF expression was already elevated by diabetes in ZDF rats. These results suggest that the use of TZDs may not increase VEGF expression in the retina of diabetic patients. In fact, several studies have suggested that the use of TZD may even have antiangiogenic effects (38 , 39) . However, the finding that RSG may enhance VEGF expression and vascular permeability in the retina of insulin-resistant ZF rats suggests that retinal functions should be examined in clinical studies on the use of TZDs in insulin resistance without the diagnosis of diabetes if clinical trials for this indication are considered.

We have also studied the role of PKC activation as a potential cause of TZD’s effect on vascular permeability since previous studies have suggested that PKC activation can disrupt epithelial and endothelial barriers by altering the phosphorylation state and the association of proteins such as occludin in tight junctions (19 , 20) . In addition, metabolic changes such as hyperglycemia and elevation of free fatty acids have been reported to activate PKC due to increases in DAG levels (40) . Our findings showed for the first time that DAG levels and probably PKC activation are increased in the microvessels of adipose tissues and the retina in response to RSG, but decreased in the monocytes. This difference in tissue response to RSG is consistent with the changes in vascular permeability. Previous studies by Isshiki et al. have also reported that TZDs can decrease DAG levels and PKC activation in the renal glomeruli of diabetic rats, supporting the concept that TZDs’ actions on DAG/PKC levels are tissue-specific (41) .

Several lines of evidence support the new findings on PKC activation as being responsible for a significant component of RSG-induced permeability, edema, and wt gain. One, PKCß isoform inhibitor, RBX, reduced RSG-induced capillary permeability in the fat and retina of Zucker lean and fatty rats, and mice, but did not affect the basal capillary permeability without RSG treatment. Two, these changes in vascular permeability corresponded in parallel with water contents of the adipose tissues and wt gains in the Zucker and VENIRKO mice, induced by RSG. Three, RSG was unable to induce increases in permeability and water content in the adipose tissues and the retina of PKCß KO mice, strongly supporting a role for PKC activation, especially the ß isoform, in vascular permeability changes induced by RSG. Indeed, this novel idea is further supported by a previous report of Nagpala et al. who showed that PKCß overexpression in cultured endothelial cells enhanced phorbol ester-induced increase in endothelial permeability (42) . However, since we only examined the PKCß isoform and its effects systemically in the present study, we cannot exclude the possibility of other PKC isoforms to be also involved and nonadipose tissue effects could have mediated the selective changes in capillary permeability in the adipose tissues (43 , 44) .

Previously, we have reported that RBX did not reduce the elevated expression of VEGF in the retina or the adipose tissue in diabetic rodents (30) . In the current study, VEGF expression was still increased in ZL and ZF rats treated with RBX and RSG, suggesting that the increases in DAG/PKC activation are mediating RSG or TZD’s effect on vascular permeability and edema probably independent of VEGF expression.

Thus, our results demonstrated that RSG and likely other structurally similar TZDs, are inducing edema partly in a tissue-specific manner by increasing vascular permeability in the adipose tissues. The mechanism of TZD’s effect is related to the increase in DAG/PKC activation in the microvessels of the adipose tissue but is not related to TZDs’ effect on improving insulin’s sensitivity in the endothelium. The changes in capillary permeability in the adipose tissue caused approximately a 10% increase in water content and wt, which can partly explain the clinical finding of TZD-induced wt gain since total fat content can account for 20 to 30% of body mass (1 , 11) . In addition, increases in capillary permeability will decrease intravascular vol and induce compensatory fluid and salt retention by the kidney, causing hemodilution and enhanced edema.

The mechanism for tissue selective effects of TZD to target adipose tissue on DAG/PKC and permeability was not determined. However, it is possible that PPAR{gamma} agonists can activate adipose tissue to induce the expression of many genes including the expression of lipoprotein lipase (LPL) (45) . Increases in LPL expression in adipose tissue will enhance its localization to the capillary endothelium, resulting in the elevation of metabolism of chylomicrons and triglyceride to fatty acids, which are transported across the endothelium to the adipocytes. Elevation of fatty acids in the endothelium of adipose tissue will activate DAG/PKC in a tissue-selective manner, leading to increases in capillary permeability, fluid retention, edema and part of the wt gain observed in patients treated with TZD.

During the active submission of our present study, a report was published by Zhang et al. showing that PPAR{gamma} null mice targeted to the collecting duct did not respond to the effects of RSG on wt gain and plasma vol expansion which may be related to reduced urinary sodium excretion (46) . However, although this recent study also used Evans blue dye, plasma vol was estimated assuming no change in vascular permeability after RSG treatment. Thus, it did not preclude the possibility, as demonstrated by our study, that PPAR{gamma} agonists can also increase capillary permeability and that PKCß is involved in edema formation and increased wt gain induced by PPAR{gamma} agonist.

Further study to substantiate this hypothesis on the role of increased LPL expression leading to TZD-induced PKC activation and fluid retention will be needed. As well, the possible involvement of other PKC isoforms than the ß isoform should be further examined. Clinically, the possibility that inhibition of PKCß isoform can ameliorate TZD’s side effects of edema, fluid retention and wt gain can be tested since RBX is being used in clinical trials for diabetic microvascular complications and may offer a novel option of a combination therapy of RBX and TZD with reduced side effects while having multiple indications for treatment of diabetes and its complications.

ACKNOWLEDGMENTS

The authors wish to acknowledge the secretary contribution of relative molecular mass, Amit Dave and David Everhart. This study was supported by grants from GSK Pharmaceutical and NIH Grant EY05110.

Received for publication July 29, 2005. Accepted for publication January 25, 2006.

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