Chi-Chang Juan

Amelioration of insulin resistance and hypertension in a fructose-fed rat model with fish oil supplementation

In type II diabetic patients, one can detect several pathologic changes including insulin resistance and hypertension. Sprague-Dawley rats fed a fructose-rich diet (group F) exhibited these characteristic abnormalities within 2 weeks and were an excellent laboratory animal model for research on insulin action and development of hypertension. Since fish oils containing omega-3 fatty acids have a beneficial effect in preventing atherosclerotic diseases, we performed repeated experiments to test the effects of fish oil supplementation in group F rats. Compared with control rats on a normal diet (group C), group F consistently developed hypertriglyceridemia without elevated plasma free fatty acid (FFA), fasting hyperinsulinemia together with fasting hyperglycemia (insulin resistance syndrome), and systolic hypertension within 3 weeks. Insulin-stimulated glucose uptake and insulin binding of adipocytes were significantly reduced. Rats fed the same high-fructose diet but supplemented with fish oil (group O) had alleviation of all of these metabolic defects and a normalized insulin sensitivity and blood pressure. beta-Cell function as shown by plasma glucose and insulin responses to oral glucose remained intact in group F and group O. The plasma endothelin-1 (ET-1) level and ET-1 binding to adipocytes were not different among the three groups. Based on these results, we suggest that dietary high fructose induced hypertriglyceridemia and insulin resistance with normal islet function, and that the induced hypertension was not associated with plasma ET-1 abnormalities and was probably caused by other undefined pathologic changes that can be prevented by dietary omega-3 fatty acids.

Protein arginine methyltransferase 5 is a potential oncoprotein that upregulates G1 cyclins/cyclin-dependent kinases and the phosphoinositide 3-kinase/AKT signaling cascade.

Increasing evidence suggests that PRMT5, a protein arginine methyltransferase, is involved in tumorigenesis. However, no systematic research has demonstrated the cell‐transforming activity of PRMT5. We investigated the involvement of PRMT5 in tumor formation. First, we showed that PRMT5 was associated with many human cancers, through statistical analysis of microarray data in the NCBI GEO database. Overexpression of ectopic PRMT5 per se or its specific shRNA enhanced or reduced cell growth under conditions of normal or low concentrations of serum, low cell density, and poor cell attachment. A stable clone that expressed exogenous PRMT5 formed tumors in nude mice, which demonstrated that PRMT5 is a potential oncoprotein. PRMT5 accelerated cell cycle progression through G1 phase and modulated regulators of G1; for example, it upregulated cyclin‐dependent kinase (CDK) 4, CDK6, and cyclins D1, D2 and E1, and inactivated retinoblastoma protein (Rb). Moreover, PRMT5 activated phosphoinositide 3‐kinase (PI3K)/AKT and suppressed c‐Jun N‐terminal kinase (JNK)/c‐Jun signaling cascades. However, only inhibition of PI3K activity, and not overexpression of JNK, blocked PRMT5‐induced cell proliferation. Further analysis of PRMT5 expression in 64 samples of human lung cancer tissues by microarray and western blot analysis revealed a tight association of PRMT5 with lung cancer. Knockdown of PRMT5 retarded cell growth of lung cancer cell lines A549 and H1299. In conclusion, to the best of our knowledge, we have characterized the cell‐transforming activity of PRMT5 and delineated its underlying mechanisms for the first time.

Overexpression of vascular endothelin-1 and endothelin: A receptors in a fructose-induced hypertensive rat model

Objective To examine the temporal relationship between hyperinsulinemia and hypertension in the fructosehypertensive rat model and to study the function of endothelin-1 (ET-1) in fructose-induced hypertension. Design Since ET-1 induces insulin resistance in conscious rats, we tested the hypothesis that both hyperinsulinemia and hypertension developed in the fructose-hypertensive rat model might be the sequelae of an elevated tissue content of ET-1 and ETA receptors. Materials and methods Systolic hypertension was induced within 3 weeks in male Sprague–Dawley rats fed on a fructose-rich diet. After continual monitoring of blood pressure and plasma insulin concentrations, the animals were killed at the end of experiment to determine plasma levels of ET-1, the contractile response of aortic rings to ET-1, and ET-1 and ETA receptor gene expressions. In a separate experiment, BQ-610 was administered to lower the effect of ET-1 in rats with fructose-induced hypertension. Results Compared with control rats given normal chow, the fructose-fed rats developed systolic hypertension after 3 weeks of the diet (127 ± 3.7 versus 110 ± 5.5 mmHg, P < 0.01) and hyperinsulinemia both before (107.1 ± 32.5 versus 48.5 ± 14.3 pmol/l, P < 0.005) and after (96.6 ± 63.7 versus 50.4 ± 5.6 pmol/l, P < 0.05) they became hypertensive. Although plasma ET-1 levels did not differ between the rat groups, aortic ring contraction–concentration curves, indicating vessel contractility in response to ET-1, were significantly greater in these rats than in controls (F1,72 = 12.34, P < 0.00077). Messenger RNA extracted from the tail arteries and blotted with both ET-1 and ETA probes showed that fructose-fed rats had greater ET-1 and ETA-receptor gene expression than control rats. Concomitant administration of BQ-610 to rats fed on a fructose diet significantly reduced the hypertension. Conclusions These findings suggest that elevated vascular expression of ET-1 and ETA receptor genes may mediate the development of hypertension and hyperinsulinemia in rats fed a fructose-rich diet.

Resistin induces monocyte-endothelial cell adhesion by increasing ICAM-1 and VCAM-1 expression in endothelial cells via p38MAPK-dependent pathway.

Resistin, firstly reported as an adipocyte‐specific hormone, is suggested to be an important link between obesity and diabetes. Recent studies have suggested an association between resistin and atherogenic processes. The adhesion of circulating monocytes to endothelial cells is a critical step in the early stages of atherosclerosis. The purpose of the present study was to investigate the effect of resistin on the adhesion of THP‐1 monocytes to human umbilical vein endothelial cells (HUVECs) and the underlying mechanism. Our results showed that resistin caused a significant increase in monocyte adhesion. In exploring the underlying mechanisms of resistin action, we found that resistin‐induced monocyte adhesion was blocked by inhibition of p38MAPK activation using SB203580 and SB202190. Furthermore, resistin increased the expression of intercellular adhesion molecule‐1 (ICAM‐1) and vascular cell adhesion molecule‐1 (VCAM‐1) by HUVECs and these effects were also p38MAPK‐dependent. Resistin‐induced monocyte adhesion was also blocked by monoclonal antibodies against ICAM‐1 and VCAM‐1. Taken together, these results show that resistin increases both the expression of ICAM‐1 and VCAM‐1 by endothelial cells and monocyte adhesion to HUVECs via p38MAPK‐dependent pathways. J. Cell. Physiol. 226: 2181–2188, 2011.

Exogenous hyperinsulinemia causes insulin resistance, hyperendothelinemia, and subsequent hypertension in rats.

In many clinical and animal studies, hypertension and insulin resistance coexist, but their mechanistic relationship is unclear. We explored the causal link between these two parameters in a rat model with chronic hyperinsulinemia induced with human insulin (1 U/d) released from subcutaneously implanted minipumps. Rats with saline minipumps served as a control. During the first experiment, plasma levels of insulin and glucose and the systolic blood pressure of the two groups were continuously monitored for 17 days. In the subsequent four experiments, rats were killed on days 10 and 13 to measure plasma endothelin-1 (ET-1) levels and the glucose transport into and insulin and ET-1 binding of isolated adipocytes. In one experiment, rats were tested for oral glucose tolerance on days 10 and 13. In another experiment, ET-1 binding to the aortic plasma membrane was also determined. The results showed that rats became hyperinsulinemic throughout the experimental period by the instillation of exogenous insulin. Hyperinsulinemic rats were consistently hypoglycemic during the first day, but they became euglycemic thereafter, indicating an insulin-resistant state. Glucose intolerance was obvious by day 10, but significant hypertension was not detected until the 11th day on insulin infusion. Compared with the saline controls, insulin-infused rats had an increase of plasma ET-1 levels but a decrease of both basal and insulin-stimulated glucose transport into adipocytes. ET-1 binding to adipocytes of the insulin-infused group was elevated significantly from day 10 through day 13. ET-1 binding to the aortic membranes, supposedly downregulated by the increased plasma ET-1 and hypertension, was similar to that found in the controls on day 13. These results imply that hyperinsulinemia in rats could lead to hypertension via the elevation of plasma ET-1 levels together with an unaltered vascular binding of ET-1, which was probably unrelated to the insulin resistance.

Laparoscopic surgery in polycystic ovary syndrome: Reproductive and metabolic effects

Polycystic ovary syndrome (PCOS) is the most common cause of chronic anovulation. Clomiphene citrate (CC) is the first-line treatment for ovulation induction for infertile women with PCOS. In CC-resistant women, a particular surgical method, laparoscopic ovarian drilling (LOD), has been proposed in recent years as an alternative treatment. LOD produces overall spontaneous ovulation and pregnancy rates of 30 to 90% and 13 to 88%, respectively, for CC-resistant PCOS women. The mechanism of LOD is still unknown. The reduction of serum androgen level is believed to be the possible mechanism of LOD to improve spontaneous ovulation and promote fertility in women with PCOS. In addition, LOD may cause a significant reduction in serum luteinizing hormone and insulin levels. However, it should be kept in mind that postoperative adhesion is the most common adverse effect of LOD, and more punctures may be responsible for premature ovarian failure.

Insulin Up-Regulates Heme Oxygenase-1 Expression in 3T3-L1 Adipocytes via PI3-Kinase- and PKC-Dependent Pathways and Heme Oxygenase-1–Associated MicroRNA Downregulation

Heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme catabolism, has antioxidant, antiinflammatory, and antiapoptotic effects in many physiological systems. HO-1 activity in obese mice is lower than in controls, and a sustained increase in HO-1 protein levels ameliorates insulin resistance and compensatory hyperinsulinemia. In the present study, we explored the regulatory effect of insulin on HO-1 expression in 3T3-L1 adipocytes and the underlying mechanism. We investigated the time- and dose-effect of insulin on HO-1 expression in 3T3-L1 adipocytes. Using specific inhibitors acting on insulin signaling pathways, we clarified the involvement of insulin downstream signaling molecules in insulin-regulated HO-1 expression. We also investigated the involvement of microRNAs (miRNAs) in insulin-regulated HO-1 expression using microarray and real-time RT-PCR assays. In an in vivo study, we performed insulin/glucose coinfusion in rats to increase circulating insulin levels for 8 h, then measured adipocyte HO-1 expression. Insulin caused a significant increase in HO-1 expression that was time- and dose-dependent, and this effect was blocked by inhibition of phosphatidylinositol 3 (PI3)-kinase activation using LY294002 (50 μM) or of protein kinase C activation using Ro-318220 (2 μM), but not by an Akt inhibitor, triciribine (10 μM). Furthermore, incubation of 3T3-L1 adipocytes with 100 nm insulin resulted in a significant decrease in levels of the miRNAs mir-155, mir-183, and mir-872, and this effect was also blocked by pretreatment with LY294002 or Ro-318220, but not triciribine. An in vivo study in rats showed that 8 h of a hyperinsulinemic euglycemic state resulted in a significant increase in adipocyte HO-1 expression. In conclusion, insulin increases HO-1 protein expression in 3T3-L1 adipocytes via PI3-kinase and protein kinase C-dependent pathways and miRNAs down-regulation.

Evidence that endothelin-1 (ET-1) inhibits insulin-stimulated glucose uptake in rat adipocytes mainly through ETA receptors

The specificity of endothelin (ET) receptors involved in the inhibition of insulin-stimulated glucose uptake (ISGU) in rat adipocytes was investigated. Adipocytes were isolated from the epididymal fat pads of Sprague-Dawley rats. To determine receptor subtypes, we used three ET isopeptides, ET-1 and ET-2, both of which are nonselective agonists, and ET-3, a selective agonist for ETC receptors, to displace [125I]ET-1 binding from the fat cells. The efficiency of displacement was ET-1 > ET-2 >> ET-3, indicating that the primary receptors involved belonged to the ETA subtype. At an equal concentration of 1 micromol/L, BQ-610, a selective ETA antagonist, displaced [125I]ET-1 from binding to fat cells, whereas IRL-1038, a selective ETB antagonist, did not. Using [3H]2-deoxy-D-1-glucose ([3H]2-DG) as a tracer in studies of glucose uptake, we found that equimolar BQ-610 completely reversed the inhibitory effect of ET-1 on ISGU, whereas IRL-1038 was ineffective. Northern blot analysis of adipocyte receptors showed abundant mRNA for ETA, but no ETB subtype. These results clearly demonstrate that ETA is the predominant receptor in rat adipocytes.

Effect of Endothelin-1 on Lipolysis in Rat Adipocytes

Objective: To explore the role of endothelin‐1 (ET‐1) on lipid metabolism, we examined the effect of ET‐1 on lipolysis in rat adipocytes.

Angiotensin II enhances endothelin-1-induced vasoconstriction through upregulating endothelin type A receptor.

Endothelin-1 (ET-1) is the most potent vasoconstrictor by binding to endothelin receptors (ETAR) in vascular smooth muscle cells (VSMCs). The complex of angiotensin II (Ang II) and Ang II type one receptor (AT1R) acts as a transient constrictor of VSMCs. The synergistic effect of ET-1 and Ang II on blood pressure has been observed in rats; however, the underlying mechanism remains unclear. We hypothesize that Ang II leads to enhancing ET-1-mediated vasoconstriction through the activation of endothelin receptor in VSMCs. The ET-1-induced vasoconstriction, ET-1 binding, and endothelin receptor expression were explored in the isolated endothelium-denuded aortae and A-10 VSMCs. Ang II pretreatment enhanced ET-1-induced vasoconstriction and ET-1 binding to the aorta. Ang II enhanced ETAR expression, but not ETBR, in aorta and increased ET-1 binding, mainly to ETAR in A-10 VSMCs. Moreover, Ang II-enhanced ETAR expression was blunted and ET-1 binding was reduced by AT1R antagonism or by inhibitors of PKC or ERK individually. In conclusion, Ang II enhances ET-1-induced vasoconstriction by upregulating ETAR expression and ET-1/ETAR binding, which may be because of the AngII/Ang II receptor pathways and the activation of PKC or ERK. These findings suggest the synergistic effect of Ang II and ET-1 on the pathogenic development of hypertension.