Yung-Pei Hsu

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 increases lipid accumulation by affecting class A scavenger receptor, CD36 and ATP-binding cassette transporter-A1 in macrophages

AIMS Resistin promotes macrophage-foam cell formation, but the mechanisms are unclear. In macrophages, lipid uptake is regulated by scavenger receptors (SR-A and CD36), while the cholesterol efflux is regulated by SR-BI, ATP-binding cassette transporter-A1 (ABCA1) and ABCG1. We investigated the mechanisms underlying the dysregulation by resistin of these regulators leading to promotion of lipid accumulation in bone marrow-derived macrophages. MAIN METHODS Western blotting, real-time PCR and oil red O staining were performed. KEY FINDINGS Resistin exacerbated lipid accumulation in oxLDL-treated macrophages. Resistin treatment of oxLDL-untreated macrophages showed increased SR-A and CD36 mRNA and protein levels, and decreased ABCA1 protein level, while having no effect on SR-BI or ABCG1 expression. Up-regulation of SR-A and CD36 by resistin resulted from activation of AP-1 and PPARgamma, respectively, and this was confirmed by the lack of activation of either after AP-1 inhibition using curcumin or SP600125, or PPARgamma inhibition using GW9662, respectively. The down-regulation of ABCA1 by resistin was not accompanied by a reduced mRNA level or an activation of LXRalpha/RXR, but resulted from enhanced protein degradation as revealed by the abolition of the down-regulation after inhibition of the proteasome pathway using ALLN or MG-132. A combined inhibition by SP600125, GW9662 and ALLN prevented resistin-induced exacerbation of lipid accumulation in oxLDL-treated macrophages. SIGNIFICANCE Resistin promotes foam cell formation via dysregulation of SR-A, CD36 and ABCA1. SR-A and CD36 are transcriptionally up-regulated by resistin through AP-1 and PPARgamma, respectively, whereas ABCA1 is down-regulated by resistin through proteasome-mediated enhancement of protein degradation.

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.

Endothelin-1 exacerbates lipid accumulation by increasing the protein degradation of the ATP-binding cassette transporter G1 in macrophages.

Endothelin‐1 (ET‐1), a potent proatherogenic vasoconstrictive peptide, is known to promote macrophage foam cell formation via mechanisms that are not fully understood. Excessive lipid accumulation in macrophages is a major hallmark during the early stages of atherosclerotic lesions. Cholesterol homeostasis is tightly regulated by scavenger receptors (SRs) and ATP‐binding cassette (ABC) transporters during the transformation of macrophage foam cells. The aim of this study was to investigate the possible mechanisms by which ET‐1 affects lipid accumulation in macrophages. Our results demonstrate that oxidized low‐density lipoprotein (oxLDL) treatment increases lipid accumulation in rat bone marrow‐derived macrophages. Combined treatment with ET‐1 and oxLDL significantly exacerbated lipid accumulation in macrophages as compared to treatment with oxLDL alone. The results of Western blotting show that ET‐1 markedly decreased the ABCG1 levels via ET type A and B receptors and activation of the phosphatidylinositol 3‐kinase pathway; however, ET‐1 had no effect on the protein expression of CD36, SR‐BI, SR‐A, or ABCA1. In addition, real‐time PCR analysis showed that ET‐1 treatment did not affect ABCG1 mRNA expression. We also found that ET‐1 decreases ABCG1 possibly due to the enhancement of the proteosome/calpain pathway‐dependent degradation of ABCG1. Moreover, ET‐1 significantly reduced the efficiency of the cholesterol efflux in macrophages. Taken together, these findings suggest that ET‐1 may impair cholesterol efflux and further exacerbate lipid accumulation during the transformation of macrophage foam cells. J. Cell. Physiol. 226: 2198–2205, 2011.

Endothelin-1 exacerbates development of hypertension and atherosclerosis in modest insulin resistant syndrome

Endothelin-1 (ET-1) is known as potent vasoconstrictor, by virtue of its mitogenic effects, and may deteriorate the process of hypertension and atherosclerosis by aggravating hyperplasia and migration in VSMCs. Our previous study demonstrated that insulin infusion caused sequential induction of hyperinsulinemia, hyperendothelinemia, insulin resistance, and then hypertension in rats. However, the underlying mechanism of ET-1 interfere insulin signaling in VSMCs remains unclear. To characterize insulin signaling during modest insulin resistant syndrome, we established and monitored rats by feeding high fructose-diet (HFD) until high blood pressure and modest insulin resistance occurred. To explore the role of ET-1/ETAR during insulin resistance, ETAR expression, ET-1 binding, and insulin signaling were investigated in the HFD-fed rats and cultured A-10 VSMCs. Results showed that high blood pressure, tunica medial wall thickening, plasma ET-1 and insulin, and accompanied with modest insulin resistance without overweight and hyperglycemia occurred in early-stage HFD-fed rats. In the endothelium-denuded aorta from HFD-fed rats, ETAR expression, but not ETBR, and ET-1 binding in aorta were increased. Moreover, decreasing of insulin-induced Akt phosphorylation and increasing of insulin-induced ERK phosphorylation were observed in aorta during modest insulin resistance. Interestingly, in ET-1 pretreated VSMCs, the increment of insulin-induced Akt phosphorylation was decreased whereas the increment of insulin-induced ERK phosphorylation was increased. In addition, insulin potentiated ET-1-induced VSMCs migration and proliferation due to increasing ET-1 binding. ETAR antagonist reversed effects of ET-1 on insulin-induced signaling and VSMCs migration and proliferation. In summary, modest insulin resistance syndrome accompanied with hyperinsulinemia leading to the potentiation on ET-1-induced actions in aortic VSMCs. ET-1 via ETAR pathway suppressed insulin-induced AKT activation, whereas remained insulin-induced ERK activation. ET-1 and insulin synergistically potentiated migration and proliferation mainly through ETAR/ERK dependent pathway, which is dominant in VSMCs during modest insulin resistance syndrome. Therefore, ET-1 and ETAR are potential targets responsible for the observed synergism effect in the hypertensive atherosclerotic process through enhancement of ET-1 binding, ET-1 binding, ETAR expression, and ET-1-induced mitogenic actions in aortic VSMCs.

Major urinary protein 1 interacts with cannabinoid receptor type 1 in fatty acid-induced hepatic insulin resistance in a mouse hepatocyte model.

Hepatic insulin resistance (HIR) is a metabolic abnormality characterized by increased gluconeogenesis which usually contributes from an elevation of free fatty acids. Cannabinoid receptor type 1 (CB1R) and major urinary protein 1 (MUP1) are thought to play pivotal roles in mitochondrial dysfunction, liver steatosis and insulin resistance. The aim of this study was to explore the role of MUP1 in CB1R-mediated HIR through the dysregulation of mitochondrial function in AML12 mouse hepatocytes challenged with high concentration of free fatty acids (HFFA). Firstly we observed that treatment of AM251, a selective CB1R antagonist, obviously reversed the HFFA-induced reduction of MUP1 protein expression both in vivo and in vitro. Additionally, our results revealed that AM251 also reverted HFFA-mediated decrease of the mRNA level of mitochondrial biogenesis-related factors, mtDNA amount, ATP production, mitochondrial respiratory complexes-I and -III, and mitochondrial membrane potential, thus consequently might correlate with a parallel reduction of ROS production. Meanwhile, AM251 attenuated HFFA-induced impairment of insulin signaling phosphorylation and elevation of phosphoenolpyrvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase), two key enzymes of gluconeogenesis. Silence of MUP1 gene abolished the inhibitory effect of AM251 on HFFA-mediated elevation of PEPCK and G6Pase expression, whereas the suppression of insulin signaling and mRNA level of mitochondrial biogenesis-related factors were only partially recovered. Altogether, these findings suggest that the anti-HIR effect of AM251 via improvement of mitochondrial functions might occur in a MUP1-dependent manner.

Using proteomics to discover novel biomarkers for fatty liver development and response to CB1R antagonist treatment in an obese mouse model.

Over activity of cannabinoid receptor type 1 (CB1R) plays a key role in increasing the incidence of obesity‐induced non‐alcoholic fatty liver disease. Tissue proteome analysis has been applied to investigate the bioinformatics regarding the mode of action and therapeutic mechanism. The aim of this study was to explore the potential pathways altered with CB1R in obesity‐induced fatty liver. Male C57BL/6 mice were fed either a standard chow diet (STD) or a high‐fat diet (HFD) with or without 1‐week treatment of CB1R inverse agonist AM251 at 5 mg/kg. Then, liver tissues were harvested for 2DE analysis and protein profiles were identified by using MALDI‐MS. Results showed that eight of significantly altered protein spots at the level of changes > twofold were overlapped among the three groups, naming major urinary protein 1, ATP synthase subunit β, glucosamine‐fructose‐6‐phosphate aminotransferase 1, zine finger protein 2, s‐adenosylmethionine synthase isoform type‐1, isocitrate dehydrogenase subunit α, epoxide hydrolase 2 and 60S acidic ribosomal protein P0. These identified proteins were involved in glucose/lipid metabolic process, xenobiotic metabolic system, and ATP synthesized process in mitochondria. Based on the findings, we speculated that CB1R blockade might exert its anti‐metabolic disorder effect via improvement of mitochondrial function in hepatic steatosis in HFD condition.

Endothelin Type A Receptor Genotype is a Determinant of Quantitative Traits of Metabolic Syndrome in Asian Hypertensive Families: A SAPPHIRe Study

Co-heritability of hypertension and insulin resistance (IR) within families not only implies genetic susceptibility may be responsible for these complex traits but also suggests a rational that biological candidate genes for hypertension may serve as markers for features of the metabolic syndrome (MetS). Thus we determined whether the T323C polymorphism (rs5333) of endothelin type A (ETA) receptor, a predominant receptor evoking potent vasoconstrictive action of endothelin-1, contributes to susceptibility to IR-associated hypertension in 1694 subjects of Chinese and Japanese origins. Blood pressures (BPs) and biochemistries were measured. Fasting insulin level, insulin-resistance homeostasis model assessment (HOMAIR) score, and area under curve of insulin concentration (AUCINS) were selected for assessing insulin sensitivity. Genotypes were obtained by methods of polymerase chain reaction-restriction fragment length polymorphism. Foremost findings were that minor allele frequency of the T323C polymorphism was noticeable lower in our overall Asian subjects compared to multi-national population reported in gene database; moreover both the genotypic and allelic frequencies of the polymorphism were significantly different between the two ethnic groups we studied. The genotype distributions at TT/TC/CC were 65, 31, 4% in Chinese and 51, 41, 8% in Japanese, respectively (p < 0.0001). Additionally, carriers of the C homozygote revealed characteristics of IR, namely significantly higher levels of fasting insulin, HOMAIR score, and AUCINS at 29.3, 35.3, and 39.3%, respectively, when compared to their counterparts with TT/TC genotypes in Chinese. Meanwhile, the CC genotype was associated with a higher level of high density lipoprotein cholesterol in Japanese. No association of the polymorphism with BP was observed. This study demonstrated for the first time that T323C polymorphism of ETA receptor gene was associated with an adverse insulin response in Chinese and a favorite atherogenic index in Japanese.