Role of GPR40 mediates the effects of FFAs on lipoapoptosis in mouse β-cell line NIT-1

AIM: To evaluate the role of G protein-coupled receptor 40 (GPR40) mediates the effects of free fatty acids (FFAs) on lipoapoptosis in mouse β-cell line NIT-1 and the mechanisms involved in this process.METHODS: NIT-1 cells were supplemented with palmitate (500 μmol/L) or oleate (500 μmol/L) for 48 h, then apoptosis of the cells was determined by the methods of Hoechst 33342, TUNEL and flow cytometry (Annexin V/PI). The small interfering RNA technique was used to inhibit the expression of GPR40 in NIT-1 cell. The mock, control siRNA and GPR40 siRNA transfected cells were either supplemented with palmitate (500 μmol/L) or co-incubated with palmitate and oleate (500 μmol/L for each) for 48 h. The percentages of apoptotic cells were quantified. The expression of p-c-Jun, Bcl-2 and Bax were detected by Western blotting.RESULTS: Palmitate induced β cell lipoapoptosis, whereas oleate inhibited NIT-1 cells from palmitate-induced lipoapoptosis. No significant difference of the percentages of apoptotic cells was indicated among the mock, control siRNA and GPR40 siRNA transfected cells treated with palmitate (P>0.05). However, after co-incubated with palmitate and oleate (500 μmol/L for each) for 48 h, the percentage of apoptotic cells in GPR40 siRNA transfected cells was greater than that in mock (P<0.05), while the expression of p-c-Jun was decreased. The expressions of Bcl-2 and Bax were not affected.CONCLUSION: Palmitate induced β cell lipoapoptosis might not be mediated through GPR40, whereas oleate inhibits NIT-1 cells from palmitate-induced lipoapoptosis, which is mediated at least in part through GPR40, the change of c-Jun expression may play an role in this process, suggesting that GPR40 might be implicated in the control of β cell mass plasticity and GPR40 probably provides a link between obesity and type 2 diabetes.     

Discovery of 20-HETE receptor and its effects on hypertension

20-Hydroxyeicosatetraenoic acid (20-HETE) is a potent vasoactive eicosanoid discovered in recent years, which has multiple vascular effects including stimulation of smooth muscle contractility, migration, and proliferation, as well as endothelial cell dysfunction and inflammation. Clinical and experimental research suggests that 20-HETE is an important mediator of cardiovascular function, and plays a critical role in the pathogenesis of hypertension, stroke, myocardial infarction and vascular diseases. Recently, G-protein-coupled receptor 75 (GPR75) was identified as a 20-HETE receptor and mediated the development of 20-HETE-dependent hypertension. The discovery of 20-HETE-GPR75 pairing provides the molecular basis for the signaling and pathophysiological functions mediated by 20-HETE in hypertension and cardiovascular diseases. In this brief review, we discuss the recent findings related to the effects of 20-HETE in the pathogenesis of hypertension and the discovery and effects of GPR75.     

Expression of urotensinⅡ receptor GPR14 in aorta of apoE knockout mice

AIM: To investigate the expression of the urotensin Ⅱ (UⅡ) receptor GPR14 in the aorta of apoE knockout mouse. METHODS: The expression of GPR14 in the aorta of apoE knockout C57BL/6J mice at various ages (18 weeks, 28 weeks, and 38 weeks old, respectively) was determined with competitive RT-PCR. A binding assay of ［125I］-UⅡ on the aortic tissue was also performed in 28 weeks group. RESULTS: We found significant upregulation of GPR14 mRNA at all three ages. Compared with wild type group at the same age, the GPR14 mRNA level in apoE knockout mice increased 54.2% in 18 week group (P<0.05), 50.0% in 28 weeks group (P<0.05) and 97.0% in 38 weeks group (P<0.01). In the knockout group or in the wild type group, expressions of GPR14 in the 28 weeks time point were significantly higher than that in other two age groups, and there was no difference between the 18 weeks and 38 weeks group. In the binding assay, the Bmax of ［125I］-UⅡ to the aorta of apoE knockout mouse at 28 weeks increased 64% compared with the wild type (P<0.01), and no difference about the Kd between the two groups was observed. CONCLUSION: UⅡ and its receptor probably play an important role in the development of atherosclerosis.     

Role of intestinal microbiota-farnesoid X receptor axis in metabolic diseases

Intestinal microbiota is associated with metabolic diseases such as obesity, nonalcoholic fatty liver disease and insulin resistance. Farnesoid X receptor (FXR), also known as bile acid receptor, is a typical nuclear receptor, which is involved in the regulation of bile acid and glycolipid metabolism. Intestinal microbiota regulates FXR activity by affecting bile acid composition, where bile acid hydrolase plays an important role. Recent studies have found that intestinal microbiota affects the development of metabolic diseases through regulating the FXR, and the intestinal microbiota-FXR axis may be an ideal drug target for metabolic diseases.     

Expression of urotensin II and its receptor in nephridial tissues of rats with acute renal damage

AIM: To observe the mRNA expression of urotensin II (UII) and its receptor (G protein-coupled receptor 14,GPR14) in nephridial tissues of rats with acute renal damage. METHODS: Male Wistar rats were divided into 2 groups: the rats in control group (n=10) were administered with normal saline by gavage; the rats in model group (n=30) were administered with Caulis Aristolochiae manshuriensis (CAM) by gavage for 25 d to induce acute renal damage. Every 5 rats in model group were sacrificed on the 3rd, 7th, 15th and 25th days during CAM treatment and all rats in the 2 groups were killed 10 d after withdraw of CAM. The kidneys were collected for pathological observation and the UII and GPR14 mRNA examination.RESULTS: The degeneration, necrosis and disintegration in tubules were observed as major pathological changes in the rat kidneys after 3 d of CAM administration. The pathological changes were aggravated following the duration of in CAM administration, and were remained and even worsen when CAM was withdrawn for 10 d. Compared with control group, the mRNA expression of UII was significantly elevated (P<0.05) at the time point of CAM administration for 15 d,even obviously increased (P<0.01) at the time point of CAM administration for 25 d, and remained at the highest levels to the end of the observation. The mRNA expression of GPR14 was significantly increased (P<0.05) at the time point of CAM administration for 7 d, became higher (P<0.01) on the 15th day, and gradually increased as the experimental time went on. CONCLUSION: The mRNA levels of UII and its receptor are significantly elevated in CAM-induced renal lesion in rats, suggesting that UII plays a pathological role in the development of acute renal damage.     

Research progress in prorenin and prorenin receptor

The renin-angiotension system (RAS) plays an important role in cardiovascular and renal physiology and diseases. Recent discoveries of prorenin and prorenin receptor add new contents to RAS. Renin and prorenin binding to the prorenin receptor not only target and facilitate angiotensin generation but also lead to activation of prorenin receptor signal transduction pathways, which is distinct from classical RAS signaling. In this paper, the construction, function and signal trasduction of prorenin, prorenin receptor and handle region peptide are reviewed.     

Toll-like receptor 4 and damage of central nervous system

As a receptor mediating the transmembrane signal transduction in the innate immunity, Toll-like receptor 4 (TLR4) is a bridge between innate immunity and required immunity, and plays an important role when signal-transducing of some cells are activated. Recent reports show that TLR4 expresses in the different glial cells and strongly links to the innate immune activation and inflammatory response in the central nervous system (CNS). TLR4 plays a key role in the processes of brain damage by infection of the CNS, stroke, cerebral hemorrhage and trauma. In this review, we concentrate on recent findings regarding the progress of function and mechanism of TLR4 in the processes of the CNS damage in various diseases.     

矮化中间砧‘宫藤富士’苹果栽植密度对树体生长、冠层光照和果实产量的影响

2011 年春季定植的矮化中间砧苹果成品苗（3 年根 1 年干的‘宫藤富士’/SH6/平邑甜茶）为试材，设置 7 种不同的栽植密度（株行距分别为 1 m × 3 m、1.5 m × 3 m、2 m × 3 m、0.75 m × 4 m、1 m × 4 m、1.25 m × 4 m 和 1.5 m × 4 m），细纺锤形整枝修剪，自栽植第 2 年，连续 7 年调查 7 种栽植密度对树体生长、冠层光照分布、果实产量和品质的影响。随着树龄的增长，不同栽植密度下树干粗度和总枝量逐年增加，不同处理间树干粗度无显著差异，第 7 年 1 m × 3 m 和 0.75 m × 4 m 两个栽植密度下树体总枝量超过 140 万条 · hm-2，第 8 年均超过 140 万条 · hm-2。栽植前期（第 2 ~ 4 年）各栽植密度树体短枝比例不断增加，长枝比例不断减少，第 5 年各栽植密度枝类组成趋于稳定；综合稳产 3 年（第 6 ~ 8 年）树体的枝类组成数据，4 m 行距的短枝比例明显高于 3 m 行距，长枝比例略低。树体冠层平均相对光照强度由高到低的株行距处理依次为 1.5 m × 4 m（63.87%）、1.25 m × 4 m（61.44%）、2 m × 3 m（61.27%）、1 m × 4 m（59.19%）、0.75 m × 4 m（55.79%）、1.5 m × 3 m（53.67%）和 1 m × 3 m（49.37%）；相同栽植株数下，4 m 行距处理低光效（相对光照强度小于 40%）的区域比例显著小于 3 m 行距。比较前 5 年的累计产量，以行距 4 m 和 1 m × 3 m 的最高。综合稳产 3 年的结果情况，大果率（单果质量 > 200 g 的果实产量占总产量的比例）以 4 m 行距和 2 m × 3 m 的最高。各栽植密度下的果实的可溶性固形物含量、固酸比、果形指数和果实硬度均无显著差异。综上，采用 4 m 行距，1 ~ 1.25 m 株距，树体成形快，稳产后树体结构合理，冠层光照充足，低效光区比例少，前期产量高。     

Mechanism of renin release and renin inhibitors

The renal local renin-angiotensin system (RAS) plays a key role in regulating the balance of water, electrolytes and blood pressure. Release of renin is the rate-limiting step of RAS activation. Recent studies have found that G-protein-coupled receptor 91 (GPR91) is highly expressed in the kidney, and can be activated by succinate, causing renin release. In recent years, from the source of renin to block the activation of RAS has become a clinical approach. In this paper, the mechanism of succinate/GPR91 to regulate renin release and application of renin inhibitors are reviewed.     

Progression in the study on roles of farnesoid X receptor

The farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily. It was studied extensively as a receptor for bile acids at first and found to control the metabolism of bile acids. In the past several years, FXR has been found that besides maintaining the homeostasis of bile acids, it also regulates the metabolism of lipid and carbohydrate, protects hepatocytes, promotes hepatic regeneration, inhibits hepatic fibrosis, regulates intestinal bacterial growth, etc. This article reviews the study progression of FXR, especially in the new discovery of FXR functions.     

4-Hydroxytamoxifen-stimulated activation of ezrin is mediated via G-protein-coupled receptor 30 and promotes human breast cancer MCF-7 cell migration

AIM：To investigate the effect of 4-hydroxytamoxifen (OHT) on the migration of human breast cancer cell line MCF-7. METHODS：The cell migration ability was assayed by scratch healing experiment. The protein expression of Src, p-Src, ezrin and p-ezrin were examined by Western blotting. RESULTS：The results of scratch healing experiment confirmed that both OHT and estradiol (E2) promoted MCF-7 cell migration and the E2-enhanced the cells migration was not inhibited by OHT. The most effective concentration of OHT that enhanced cell migration was 5 μmol/L. Significant promotion of the cell migration was observed at 6 h after OHT treatment. Increased p-ezrin and p-Src expression was observed after treatment with OHT and G-protein-coupled receptor 30 (GPR30) agonists G1. The expression of p-ezrin after OHT treatment was inhibited by G15 (GPR30 blocker) or PP2（Src inhibitor）. CONCLUSION：4-Hydroxytamoxifen promotes MCF-7 cell migration by activation of ezrin via GPR30 and c-Src.     

Oxytocin receptor and tumor

Oxytocin receptor (OTR) is a member of G-protein coupled receptor, which has been found in many tumors and cancer cell lines. Many studies revealed oxytocin (OT) may inhibit the tumor and cancer cell growth and proliferation, but the mechanism of this inhibition is not wel known. Some experiments indicated cAMP-PKA system participates in the signal transduction that oxytocin inhibits the cancer cell proliferation. However, other experiments showed the signal transduction for oxytocin in the Hs578T carcinosacoma cel is the same as that in the normal cells. In this review, the relationship between OTR and tumors are summarized.     

Regulation of AMPK/PPARα/SCAD signal pathway on cardiac hypertrophy

AIM：To study the effect of short-chain acyl-coenzyme A dehydrogenase (SCAD）on cardiac hypertrophy and to explore the role of adenosine monophosphate-activated protein kinase (AMPK)/peroxisome proliferator-activated receptor α (PPARα） signal pathway in the regulation of SCAD during the development of cardiac hypertrophy. METHODS：The optimal sequence of SCAD interference was chosen by Western blotting and real-time PCR. The cardiomyocytes were treated with fenofibrate (10 μmol/L) for 24 h and subsequently stimulated with the optimal sequence of SCAD interference. The changes of SCAD expression at mRNA and protein levels, the enzyme activity of SCAD, the cardiomyocyte surface area and free fatty acids were determined. Using real-time PCR for analyzing the markers of cardiac hypertrophy, the mRNA expression of atrial natriuretic factor (ANF） and brain natriuretic peptide (BNP) was detected to judge the development of cardiac hypertrophy. The cardiomyocytes were treated with fenofibrate (10 μmol/L) or AMPK activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR, 0.5 mmol/L) for 30 min and subsequently stimulated with phenylephrine (PE, 20 μmol/L) for 24 h. The changes of cardiomyocyte surface area, free fatty acids, and the expression of SCAD, PPARα and p-AMPKα (T172) at mRNA and protein levels were observed. RESULTS：The effect of optimal sequence siRNA-1186 and PE on the cardiomyocytes was the same. Compared with control group, the expression of ANF and BNP at mRNA level, the cardiomyocyte surface area and free fatty acids were increased obviously in siRNA-1186 group. After pretreated with fenofibrate (10 μmol/L), the expression of PPARα and SCAD, and the enzyme activity of SCAD were significantly increased, while the free fatty acids were decreased, indicating that fenofibrate prevented the development of cardiac hypertrophy induced by knockdown of SCAD. Compared with control group, the expression of SCAD, PPARα and p-AMPKα (T172) at mRNA and protein levels was significantly down-regulated, and the enzyme activity of SCAD was obviously decreased in PE group. Compared with PE group, the expression of SCAD, PPARα and p-AMPKα (T172) was significantly up-regulated, and the cardiomyocyte surface area and the content of free fatty acids were obviously decreased in the cardiomyocytes pretreated with fenofibrate or AICAR for 30 min. CONCLUSION：Down-regulation of SCAD is related to the cardiac hypertrophy and energy metabolism. AMPK/PPARα/SCAD signaling pathway may regulate cardiac hypertrophy directly.     

mRNA expression of insulin receptor and leptin receptor in liver of nonalcoholic fatty liver disease from diabetic rats

AIM: To observe the pathologic changes of liver in diabetic rats and to investigate the role of mRNA expression of insulin receptor and leptin receptor in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). METHODS: Twenty male Sprague-Dawley rats were divided randomly into two groups: normal control group and diabetic group. After fed with high-fat diet for 4 weeks, diabetic rats were injected with streptozotocin at a dosage of 30 mg/kg intraperitoneally to induce NAFLD model of type 2 diabetes mellitus. Then the diabetic animals were fed with high-fat diet continuously for 12 weeks. At the end of the experiment, the rats were sacrificed, the concentrations of blood glucose, serum lipid, ALT and AST were measured biochemically. The levels of serum leptin and serum insulin were detected by enzyme-linked immunosorbent assay (ELISA) and radio immunoassay (RIA), respectively. The pathologic changes of liver were observed under light microscopy (LM) stained with HE, Sudan Ⅲ and Masson trichrome staining, respectively. The ultra-structural changes of liver were observed under transmission electron microscopy (TEM). Additionally, the mRNA expressions of PEPCK, G6Pase, insulin R and leptin R from rat livers were assayed by semi-quantitative RT-PCR. RESULTS: The levels of blood glucose, serum insulin, serum TG, ALT and AST increased significantly (P<0.01), serum TC elevated (P<0.05), and the levels of serum leptin decreased (P<0.01) in diabetic group compared to those in normal control group. Obvious liver fatty degeneration, piecemeal necrosis with accompanying inflammatory infiltration and fibrosis were found under LM. Hepatocytes pyknosis, lots of lipid deposits in cytoplasm of hepatocytes, proliferation of collagen in space of Disse were observed under TEM in diabetic group. The expression of insulin R and leptin R mRNA in liver from diabetic rats increased significantly (P<0.01) while the expression of PEPCK and G6Pase mRNA remained unchanged. CONCLUSION: Insulin resistance plays an important role in the pathogenesis of NAFLD. Low level of serum leptin, up-regulation of mRNA expression of insulin R and leptin R in liver caused by insulin resistance may be involved in this process.     

Alteration of serum free fatty acids during the carbon tetrachloride-induced fatty liver in rats

AIM: To investigate the alteration of serum free fatty acids in the carbon tetrachloride-induced fatty liver. METHODS: Drug-induced fatty liver rat models were built by injection 40% CCl₄. Serum free fatty acids were analyzed by gas chromatography. RESULTS: In the composition of serun free fatty acids, polyunsaturated fatty acids[oleic acid C18:1,(28.672±7.332/mg·L^-1 vs 41.373±2.180/mg·L^-1),linoleic acid C18:2(16.739±0.871/mg·L^-1 vs 24.959±5.325/mg·L^-1),arachidonic acid C20:4(6.105±2.656/mg·L^-1 vs 9.802±0.779/mg·L^-1),P<0 05], were decreased significantly, but saturated fatty acids [lauric acid C12：0(3.368±0.330/mg·L^-1 vs 2.742 0.351/mg·L^-1),myrist ic acid C14:0(27.136 3.158/mg·L^-1 vs 16.152±0.638/mg·L^-1),palmitic acid C16:0(51.731±9.561/mg·L^-1 vs 34.522±1.401/mg·L^-1),P<0 05] increased. CONCLUSION: Serum polyunsaturated fatty acids were decreased and saturated fatty acids increased in the drug-induced fatty liver.     

Expression of peroxisome proliferator-activated receptor α in rat fatty liver

AIM:To investigate the role of expression of peroxisome proliferator-activated receptor α(PPAR α) in pathogenesis of rat fatty liver.METHODS:The rats were treated with a low dose of carbon terachloride (CCl₄) and fed a high fat diet to produce fatty liver. We determined the concentrations of triglyceride (TG), total cholesterol (TC), free fatty acid (FFA) in liver and the alanine aminotransferase (ALT) activity, tumor necrosis factor-α (TNF-α), FFA in serum and the degree of hepatocytic steatosis. Total RNA of liver was extracted, and the expression of PPAR α were analyzed by semi-quantitative RT-PCR method.RESULTS:In model group, the hepatocytic PPAR α mRNA expression decreased to 0.41±0.28, compared to 1.41±0.29 in the control group (P＜0.01). The contents of TG, TC, FFA in model rat liver were (1.88±0.20) mmol·L^-1, (11.03±1.12) mmol·L^-1 and (1 260.38±151.27) μmol·L^-1, respectively, compared to (0.53±0.10) mmol·L^-1, (1.25±0.25) mmol·L^-1 and (334.30±27.09) μmol·L^-1 in the control group (P＜0.01). The activity of ALT, concentrations of TNF-α and FFA in serum were also increased remarkably in model group.CONCLUSION:Oxidation of fatty acid and utilization of lipids in liver are affected by reducing the expression of PPAR α, which result lipid accumulation in liver.     

Role of G-protein-coupled receptor kinases in cell migration and signal transduction

G-protein-coupled receptor kinases (GRKs) are a family of serine/threonine protein kinases. The investigators pay much attention to the roles of GRKs in the signal transduction through G-protein-coupled receptors (GPCRs) with arrestin ever since a long time ago. Due to the physiological and pathological observations with the methods of deletion or overexpression, GRKs are considered as new drug targets. The kinases play a role in the pathogenesis of hypertension and cell migration through GPCRs and Hedgehog signaling pathways. As the development of research techniques, especially bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET), the special mechanism of GRKs for GPCRs is more evident. In this review, we discuss the recent achievement in the roles of GRKs signaling and the related newest research techniques.     

Effects of short-chain acyl-CoA dehydrogenase on cardiomyocyte apoptosis

AIM: To investigate the change of short-chain acyl-CoA dehydrogenase(SCAD) expression during cardiomyocyte apoptosis and to explore the relationship between SCAD and cardiomyocyte apoptosis.METHODS: The neonatal rat cardiomyocytes treated by tert-butyl hydroperoxide(tBHP) were used as the model of cardiomyocyte apoptosis. The cell viability, the expression of SCAD at mRNA and protein levels, the activity of SCAD and the content of free fatty acids were determined.RESULTS: The mRNA and protein expression of SCAD decreased in the cardiomyocyte apoptosis model. Compared with negative control group, SCAD expression and activity were both significantly decreased in siRNA-1186 group, but the content of free fatty acids were obviously increased in the cardiomyocytes. Meanwhile, SCAD siRNA treatment triggered the same apoptosis as cardiomyocytes treated with tBHP.CONCLUSION: Down-regulation of SCAD may play an important role in primary cardiomyocyte apoptosis. Increase in the expression of SCAD may become an important part in intervening cardiomyocyte apoptosis.     

Hypoxic tubulointerstitial fibrosis and swimming exercise change expression of urotensin II and its receptor GPR14 in rats

AIM: To observe the expression of urotensin II (UII) and its receptor GPR14 in rats with hypoxic tubulointerstitial fibrosis, and to explore the changes after swimming exercise. METHODS: The animal model of hypoxic renal interstitial fibrosis was established by exposing the rats to isobaric hypoxic chamber for 7 weeks (8 h/d, 7 d/week). Forty-five male SD rats were randomly divided into normal control group (control), hypoxic 7-week group (hypoxia) and hypoxic 3-week and swimming without loads 4-week group (swimming). Serum creatinine (Scr) and blood urea nitrogen (BUN) were measured by chemical colorimetry, and UII was detected by ELISA. The content of hydroxyproline (Hyp) in the renal homogenate was assayed. The mRNA expression of UII and GPR14 were detected by RT-PCR. The protein level of UII was determined by the method of immunohistochemistry. Meanwhile, the renal specimens were prepared to observe renal interstitial fibrosis by van Gieson(VG) staining. RESULTS: The content of Scr and BUN in hypoxia group was lower than that in control group by 18.5% and 14.1%,respectively, while there was no significant difference between swimming group and hypoxia group. The content of Hyp in hypoxia group was 42.9% higher than that in control group, while swimming group was 26.1% lower than that in hypoxia group. The plasma content of UII in hypoxia group was 380.8% higher than that in control group, while swimming group was 42.6% lower than that in hypoxia group. The mRNA expression of UII in the kidneys was obviously up-regulated by 104.5% in hypoxia group compared with control group, while it was markedly down-regulated by 33.2% in swimming group compared with hypoxia group. The mRNA expression of GPR14 in the kidneys was significantly up-regulated by 35.4% in hypoxia group compared with control group. However, no significant difference between hypoxia group and swimming group was observed. The UII protein level in the kidneys of hypoxia rats was distinctly higher than that in control group and lower in swimming group than that in hypoxia group. VG staining revealed that the renal interstitial fibrosis was found in hypoxia group, which was significantly alleviated by swimming exercise. CONCLUSION: The levels of UII and GPR14 increase in the kidneys of the rats with hypoxic tubulointerstitial fibrosis. Moderate swimming exercise alleviates the tubulointerstitial fibrosis induced by hypoxia and decreases the expression of UII.