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agonist’s effects on edema and weight gain
Research Division, Joslin Diabetes Center, Harvard Medical School One Joslin Place, Boston, Massachusetts, USA
1Correspondence: Joslin Diabetes Center, One Joslin Pl., Boston, MA 02215, USA. E-mail: george.king{at}joslin.harvard.edu
SPECIFIC AIMS
In this study, we investigated the underlying mechanisms that cause the serious adverse effects of PPAR
agonists (rosiglitazone, RSG)—edema and wt gain—in models of diabetes and insulin resistance with the following aims:
1. To explore the potential involvement of protein kinase C (PKC) and vascular endothelial growth factor (VEGF), two well-established vascular permeability factors, on TZD-induced edema;
2. To examine the role of insulin’s effects on the endothelial cells on the regulation of capillary permeability.
PRINCIPAL FINDINGS
1. RSG effects on wt gain and vascular permeability
Four weeks of RSG treatment significantly increased wt gain in Zucker rats (lean: ZL, fatty: ZF, diabetic fatty: ZDF) (P<0.05 vs. untreated groups). Permeability of Evans blue (EB) dye across the endothelial barrier was examined in epididymal and perirenal fat, retina, heart, and skeletal muscle of Zucker rats. RSG treatment significantly increased vascular permeability in the epididymal fat by 30, 50, and 20% in ZL, ZF, and ZDF, respectively (P<0.05 vs. untreated groups). EB dye leakage was significantly increased in the retina of RSG-treated ZL (by 30%) and ZF rats (by 40%), but not in ZDF rats. RSG did not alter EB dye permeability in the myocardium or soleus muscle. Water content was also increased by RSG in the epididymal fat by 30, 30, and 10% in ZL, ZF, and ZDF rats, respectively, vs. their controls (P<0.05). RSG increased water content in the retina of ZF rats vs. the control (P=0.05), whereas no changes were induced by RSG in ZL and ZDF groups. Similar to EB permeability, RSG did not cause any significant changes in water content/wet wt of the heart and soleus muscle. Thus, PPAR agonists induce vascular permeability selectively in the adipose tissue.
2. RSG effects on VEGF expression
RSG treatment increased VEGF mRNA expression in adipose tissue by 1.4-, 2.2-, and 2-fold for ZL, ZF, and ZDF, respectively (P<0.05 vs. controls). RSG significantly increased VEGF expression in the retina of ZL and ZF rats but the increase in VEGF expression in ZDF rats did not reach significance. RSG did not significantly change VEGF expression in the myocardium or soleus muscle. Thus, PPAR agonists increase the expression of VEGF selectively in adipose tissues and retina.
3. RSG effects on diacylglycerol (DAG) levels and PKC activity
In adipose tissue microvessels, RSG treatment increased DAG levels in both ZL (by 65%, P=0.04) and ZF (by 70%, P=0.05) rats. RSG increased PKC activities in the membrane fractions (activated pool) in ZL (by 45%, P =0.08) and ZF (by 35%, P=0.07) rats. For the retina, RSG increased in membranous PKC activities in ZL (P=0.06) and ZF (P<0.05) rats vs. their controls. In contrast, RSG decreased PKC activity in monocytes from ZL and ZF rats.
4. Effects of PKCßbeta; inhibition on RSG effects
The addition of ruboxistaurin (RBX), a PKCßbeta; specific inhibitor, in the last 2 wk of the 4-wk RSG treatment significantly decreased EB dye leakage compared with RSG alone in the epididymal fat of both ZL and ZF rats (P<0.05, Fig. 1
A). Similar trends were observed in the retina where combined treatment with RBX decreased EB dye leakage by 20 and 25% in RSG-treated ZL and ZF rats, respectively. RBX alone did not affect basal EB dye leakage without RSG treatment in the retina, epididymal fat, or subcutaneous (s.c.) fat. Total water content in the epididymal fat of ZL and ZF rats was 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. 1B
). No significant changes were induced by RBX in the water content of retina, heart, or soleus muscle in ZL or ZF rats. ZL and ZF rats treated with RSG alone for 4 wk had increased wt gains vs. untreated ZL (P=0.001) and ZF (P<0.001). 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. 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. RBX treatments alone did not affect body wt as compared with control animals. Besides total wt, RSG 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 with RSG alone for ZL (P=0.05) and ZF rats (P=0.04). RBX did not decrease VEGF mRNA expression significantly in the adipose tissue of either ZL or ZF rats. Similar findings were obtained in the retina. Thus, PKCßbeta; inhibitors prevent edema and wt gain induced by PPAR agonists in lean and insulin-resistant Zucker rats but do not decrease VEGF expression.
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5. RSG effects in PKCßbeta; knockout (KO) mice (Fig. 2 )
To confirm the role of PKCßbeta; isoform in RSG-induced increases in vascular permeability, the effect of RSG was studied in PKCßbeta; KO mice. Permeability of EB dye was similar in wild-type (WT) and PKCßbeta; 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 PKCßbeta; KO mice. Weight gain in WT mice treated with RSG was significantly greater than in untreated WT mice (P=0.02). RSG did not induce wt gain of PKCßbeta; KO mice. Thus, PPAR agonists cannot induce edema and increase wt gain in PKCßbeta; KO mice.
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6. RSG effects in vascular endothelium specific insulin receptor knockout (VENIRKO) mice
The effects of RSG on EB dye permeability were examined in VENIRKO mice. RSG treatment increased EB dye permeability in the epididymal and s.c. fat 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<0.05 in epididymal fat for both control and VENIRKO). Similar to the Zucker rats, RSG 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. wt gains of VENIRKO and WT mice treated with RSG were increased significantly vs. untreated groups (P<0.05 for both) and higher than the combined RBX and RSG-treated group (P=0.05 in WT mice).
CONCLUSIONS AND SIGNIFICANCE
RSG increases vascular permeability selectively in the adipose tissues and retina, but not in the myocardium and skeletal muscle. The decreases in permeability observed in the adipose tissues from insulin-resistant states are new findings that 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. Further studies are needed to clarify the molecular mechanism and biological significance of the decrease in basal capillary permeability in the retina and adipose tissues in insulin-resistant states.
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’s effects on endothelial cells and insulin resistance are not essential for TZD to induce edema.
It is not clear from our results whether the increases in VEGF expression play a significant role in TZDs’ induction of edema and capillary permeability. RBX treatment decreased permeability, edema and wt gain, but did not reduce VEGF expression in the adipose tissue. Thus, TZDs may also stimulate edema due to VEGF independent pathways.
A role for PKC activation in TZDs’ effect on vascular permeability is implicated by previous studies showing 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. Additionally, metabolic changes such as hyperglycemia and elevation of free fatty acids have been reported to activate PKC due to increases in DAG levels. Our finding showed 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. Moreover, previous studies have reported that TZDs can decrease DAG levels and PKC activation in the renal glomeruli of diabetic rats, suggesting that TZDs’ actions on DAG/PKC levels are also tissue-specific.
Several lines of evidence support PKC activation as being responsible for a significant component of RSG-induced permeability, edema, and wt gain. First, PKCßbeta; isoform inhibitor RBX, reduced RSG-induced capillary permeability in the fat and retina of Zucker lean and fatty rats, and mice, but did not alter basal capillary permeability in these tissues without RSG treatment. Secondly, these changes in vascular permeability corresponded with water contents of the adipose tissues and wt gains induced by RSG in the Zucker and VENIRKO mice. Finally, RSG was unable to induce increases in permeability and water content in the adipose tissues and the retina of PKCßbeta; KO mice, strongly supporting a role for PKC activation, especially the ßbeta; isoform, in vascular permeability changes induced by RSG. either locally or via systemic effects. 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 TZDs’ effect on vascular permeability and edema, probably independent of VEGF expression.
The mechanism of TZDs’ effect may be 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 sensitivity in the endothelium. The mechanisms for edema and wt gain induced by PPAR agonists appear to be multiple. They may be related to both renal and peripheral actions of PPAR agonists. Our study suggests a mechanism by which PPAR agonists can cause edema by increasing capillary permeability selectively in adipose tissue through PKCßbeta; isoform activation.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4617fje
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  L. Q. Shen, A. Child, G. M. Weber, J. Folkman, and L. P. Aiello Rosiglitazone and Delayed Onset of Proliferative Diabetic Retinopathy Arch Ophthalmol, June 1, 2008; 126(6): 793 - 799. [Abstract] [Full Text] [PDF]
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