Intermittent protein restriction before but not after the onset of diabetic kidney disease attenuates disease progression in mice.

Background: High dietary protein intake exacerbates proteinuria in individuals with diabetic kidney disease (DKD). However, studies on the impacts of low protein diet (LPD) on DKD have yielded conflicting results. Furthermore, patient compliance to continuous protein restriction is challenging. Objective: The current study aims to investigate the effects of intermittent protein restriction (IPR) on disease progression of DKD. Methods: Diabetic KK-Ay mice were used in this study. For the IPR treatment, three consecutive days of LPD were followed by four consecutive days of normal protein diet (NPD) within each week. For early intervention, mice received IPR before DKD onset. For late intervention, mice received IPR after DKD onset. In both experiments, age-matched mice fed continuous NPD served as the control group. Kidney morphology, structure and function of mice in different groups were examined. Results: Intermittent protein restriction before DKD onset ameliorated pathological changes in kidney, including nephromegaly, glomerular hyperfiltration, tubular injuries and proteinuria, without improving glycemic control. Meanwhile, IPR initiated after DKD onset showed no renoprotective effects despite improved glucose homeostasis. Conclusion: Intermittent protein restriction before rather than after DKD onset protects kidneys, and the impacts of IPR on the kidneys are independent of glycemic control. IPR shows promise as an effective strategy for managing DKD and improving patient compliance.

Correlation between newborn weight and serum BCAAs in pregnant women with diabetes.

BACKGROUND: Branched-chain amino acids (BCAAs), including leucine, isoleucine, and valine, are essential amino acids for mammals. Maternal BCAAs during pregnancy have been associated with newborn development. Meanwhile, BCAAs have been tightly linked with insulin resistance and diabetes in recent years. Diabetes in pregnancy is a common metabolic disorder. The current study aims to assess the circulating BCAA levels in pregnant women with diabetes and their relationship with neonatal development. METHODS: The serum concentrations of BCAAs and their corresponding branched-chain α-keto acids (BCKAs) catabolites in 33 pregnant women with normal glucose tolerance, 16 pregnant women with type 2 diabetes before pregnancy (PDGM), and 15 pregnant women with gestational diabetes mellitus (GDM) were determined using a liquid chromatography system coupled to a mass spectrometer. The data were tested for normal distribution and homogeneity of variance before statistical analysis. Correlations were computed with the Pearson correlation coefficient. RESULTS: The maternal serum BCAAs and BCKAs levels during late pregnancy were higher in women with PGDM than those in healthy women. Meanwhile, the circulating BCAAs and BCKAs showed no significant changes in women with GDM compared with those in healthy pregnant women. Furthermore, the circulating BCAA and BCKA levels in women with PGDM were positively correlated with the weight of the newborn. The circulating leucine level in women with GDM was positively correlated with the weight of the newborn. BCAA and BCKA levels in healthy pregnant women showed no correlation with newborn weight. CONCLUSIONS: The serum BCAAs in pregnant women with diabetes, which was elevated in PGDM but not GDM, were positively correlated with newborn weight. These findings highlight potential approaches for early identification of high-risk individuals and interventions to reduce the risk of adverse pregnancy outcomes.

Transport of glycinin, the major soybean allergen, across intestinal epithelial IPEC-J2 cell monolayers.

Soybean allergen entering the body is the initial step to trigger intestinal allergic response. However, it remains unclear how glycinin, the major soybean allergen, is transported through the intestinal mucosal barrier. The objective of this study was to elucidate the pathway and mechanism of glycinin hydrolysate transport through the intestinal epithelial barrier using IPEC-J2 cell model. Purified glycinin was digested by in vitro static digestion model. The pathway and mechanism of glycinin hydrolysates transport through intestinal epithelial cells were investigated by cellular transcytosis assay, cellular uptake assay, immunoelectron microscopy and endocytosis inhibition assay. The glycinin hydrolysates were transported across IPEC-J2 cell monolayers in a time/dose-dependent manner following the Michaelis equation. Immunoelectron microscopy showed a number of glycinin hydrolysates appeared in the cytoplasm, but no glycinin hydrolysates were observed in the intercellular space of IPEC-J2 cells. The inhibitors, colchicine, chlorpromazine and methyl-ß-cyclodextrin, significantly inhibited the cellular uptake of glycinin hydrolysates. The glycinin hydrolysates crossed IPEC-J2 cell monolayers through the transcellular pathway. Both clathrin- and caveolae-dependent endocytosis were involved in the epithelial uptake of the hydrolysates. These findings provided potential targets for the prevention and treatment of soybean allergy.

ZBTB20 Regulates SERCA2a Activity and Myocardial Contractility Through Phospholamban.

BACKGROUND: Intracellular Ca2+ cycling determines myocardial contraction and relaxation in response to physiological demands. SERCA2a (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 2a) is responsible for the sequestration of cytosolic Ca2+ into intracellular stores during cardiac relaxation, and its activity is reversibly inhibited by PLN (phospholamban). However, the regulatory hierarchy of SERCA2a activity remains unclear. METHODS: Cardiomyocyte-specific ZBTB20 knockout mice were generated by crossing ZBTB20flox mice with Myh6-Cre mice. Echocardiography, blood pressure measurements, Langendorff perfusion, histological analysis and immunohistochemistry, quantitative reverse transcription-PCR, Western blot analysis, electrophysiological measurements, and chromatin immunoprecipitation assay were performed to clarify the phenotype and elucidate the molecular mechanisms. RESULTS: Specific ablation of ZBTB20 in cardiomyocyte led to a significant increase in basal myocardial contractile parameters both in vivo and in vitro, accompanied by an impairment in cardiac reserve and exercise capacity. Moreover, the cardiomyocytes lacking ZBTB20 showed an increase in sarcoplasmic reticular Ca2+ content and exhibited a remarkable enhancement in both SERCA2a activity and electrically stimulated contraction. Mechanistically, PLN expression was dramatically reduced in cardiomyocytes at the mRNA and protein levels by ZBTB20 deletion or silencing, and PLN overexpression could largely restore the basal contractility in ZBTB20-deficient cardiomyocytes. CONCLUSIONS: These data point to ZBTB20 as a fine-tuning modulator of PLN expression and SERCA2a activity, thereby offering new perspective on the regulation of basal contractility in the mammalian heart.

Serum branched chain amino acids: an effective indicator of diabetic kidney disease.

Introduction: In recent years, there has been a growing association between elevated circulating levels of branched-chain amino acids (BCAA) and diabetes mellitus. However, the relationship between serum BCAA levels and diabetic kidney disease (DKD) remains ambiguous. This study aims to investigate serum BCAA levels in DKD patients at various stages and assess the correlation between BCAA and clinical characteristics. Materials and methods: We enrolled patients with type 2 diabetes mellitus (T2DM) who were admitted to our hospital and categorized them into three groups based on different DKD stages: normal proteinuria, microproteinuria, and macroalbuminuria groups. Forty healthy volunteers were included as the control group, and we measured serum BCAA concentrations using liquid chromatography-mass spectrometry (LC-MS). Subsequently, we conducted correlation and regression analyses to assess the associations between BCAA and clinical indicators. Results: Serum BCAA levels were significantly elevated in T2DM patients compared to healthy controls. However, these levels exhibited a gradual decline with the progression of DKD. Furthermore, after adjusting for age, gender, and disease duration, we observed an independent association between serum albumin, urinary transferrin, and urinary microalbumin with BCAA. Discussion: Our findings suggest a noteworthy decline in serum BCAA levels alongside the advancement of DKD. Additionally, serum BCAA exhibits an independent correlation with renal function indicators. These observations point to the possibility that serum BCAA concentrations in individuals with T2DM hold promise as a crucial predictor for both the initiation and progression of DKD.

The effect of hypothyroidism on the risk of diabetes and its microvascular complications: a Mendelian randomization study.

Context: Several observational studies have found that hypothyroidism is associated with diabetes and its microvascular complications. However, the cause and effect have not been clarified. Objective: The aim of the study was to examine the causality of such associations by a Mendelian randomization study. Methods: Two-sample Mendelian randomization analysis was conducted to investigate the associations. Summary statistics for hypothyroidism were from the UK Biobank, and diabetes and its microvascular complications were from the largest available genome-wide association studies. MR-Egger, weighted median, inverse variance weighted, simple mode and weighted mode were used to examine the causal associations, and several sensitivity analyses were used to assess pleiotropy. Results: Inverse variance weighted estimates suggested that hypothyroidism was associated with type 1 diabetes and type 1 diabetes with renal complications (ß= 9.059926, se= 1.762903, P = 2.76E-07 and ß= 10.18375, se= 2.021879, P = 4.73E-07, respectively) but not type 2 diabetes and type 2 diabetes with renal complications. In addition, hypothyroidism was positively associated with severe nonproliferative diabetic retinopathy and proliferative diabetic retinopathy (ß= 8.427943, se= 2.142493, P = 8.36E-05 and ß= 3.100939, se= 0.74956, P=3.52E-05, respectively). Conclusions: The study identified the causal roles of hypothyroidism in diabetes and its microvascular complications. Hypothyroidism can lead to type 1 diabetes, type 1 diabetes with renal complications, severe nonproliferative diabetic retinopathy and proliferative diabetic retinopathy.

Targeting BCAA metabolism to potentiate metformin's therapeutic efficacy in the treatment of diabetes in mice.

AIMS/HYPOTHESIS: An increasing body of evidence has shown that the catabolism of branched-chain amino acids (BCAAs; leucine, isoleucine and valine) is impaired in obese animals and humans, contributing to the development of insulin resistance and type 2 diabetes. Promoting BCAA catabolism benefits glycaemic control. It remains unclear whether BCAA catabolism plays a role in the therapeutic efficacy of currently used glucose-lowering drugs such as metformin. METHODS: Mice were treated with vehicle or metformin (250 mg/kg per day) for more than 4 weeks to investigate the effects of metformin in vivo. In vitro, primary mouse hepatocytes and HepG2 cells were treated with 2 mmol/l metformin. The therapeutic efficacy of metformin in the treatment of type 2 diabetes was assessed in genetically obese (ob/ob) mice and high-fat-diet-induced obese (DIO) mice. Enhancing BCAA catabolism was achieved with a pharmacological agent, 3,6-dichlorobenzo[b]thiophene-2-carboxylic acid (BT2). The ob/ob mice were treated with a low-BCAA diet or intermittent protein restriction (IPR) to reduce BCAA nutritional intake. RESULTS: Metformin unexpectedly inhibited the catabolism of BCAAs in obese mice, resulting in an elevation of BCAA abundance. AMP-activated protein kinase (AMPK) mediated the impact of metformin on BCAA catabolism in hepatocytes. Importantly, enhancing BCAA catabolism via a pharmacological agent BT2 significantly potentiated the glucose-lowering effect of metformin while decreasing circulating BCAA levels in ob/ob and DIO mice. Similar outcomes were achieved by a nutritional approach of reducing BCAA intake. IPR also effectively reduced the circulating BCAA abundance and enhanced metformin's glucose-lowering effect in ob/ob mice. BT2 and IPR treatments reduced the expression of fructose-1,6-bisphosphatase 1, a rate-limiting enzyme in gluconeogenesis, in the kidney but not liver, indicating the involvement of renal gluconeogenesis. CONCLUSIONS/INTERPRETATION: Metformin self-limits its therapeutic efficacy in the treatment of type 2 diabetes by triggering the suppression of BCAA catabolism. Enhancing BCAA catabolism pharmacologically or reducing BCAA intake nutritionally potentiates the glucose-lowering effect of metformin. These data highlight the nutritional impact of protein on metformin's therapeutic efficacy and provide new strategies targeting BCAA metabolism to improve metformin's effects on the clinical outcome in diabetes.

Hepatic but not Intestinal FBP1 Is Required for Fructose Metabolism and Tolerance.

Fructose intolerance in mammals is caused by defects in fructose absorption and metabolism. Fructose-1,6-bisphosphatase 1 (FBP1) is a key enzyme in gluconeogenesis, and its deficiency results in hypoglycemia as well as intolerance to fructose. However, the mechanism about fructose intolerance caused by FBP1 deficiency has not been fully elucidated. Here, we demonstrate that hepatic but not intestinal FBP1 is required for fructose metabolism and tolerance. We generated inducible knockout mouse models specifically lacking FBP1 in adult intestine or liver. Intestine-specific deletion of Fbp1 in adult mice does not compromise fructose tolerance, as evidenced by no significant body weight loss, food intake reduction, or morphological changes of the small intestine during 4 weeks of exposure to a high-fructose diet. By contrast, liver-specific deletion of Fbp1 in adult mice leads to fructose intolerance, as manifested by substantial weight loss, hepatomegaly, and liver injury after exposure to a high-fructose diet. Notably, the fructose metabolite fructose-1-phosphate is accumulated in FBP1-deficient liver after fructose challenge, which indicates a defect of fructolysis, probably due to competitive inhibition by fructose-1,6-bisphosphate and may account for the fructose intolerance. In conclusion, these data have clarified the essential role of hepatic but not intestinal FBP1 in fructose metabolism and tolerance.

The zinc finger and BTB domain containing protein ZBTB20 regulates plasma triglyceride metabolism by repressing lipoprotein lipase gene transcription in hepatocytes.

BACKGROUND AND AIMS: Lipoprotein lipase (LPL) is responsible for the lipolytic processing of triglyceride-rich lipoproteins, the deficiency of which causes severe hypertriglyceridemia. Liver LPL expression is high in suckling rodents but relatively low at adulthood. However, the regulatory mechanism and functional significance of liver LPL expression are incompletely understood. We have established the zinc finger protein ZBTB20 as a critical factor for hepatic lipogenesis. Here, we evaluated the role of ZBTB20 in regulating liver Lpl gene transcription and plasma triglyceride metabolism. APPROACH AND RESULTS: Hepatocyte-specific inactivation of ZBTB20 in mice led to a remarkable increase in LPL expression at the mRNA and protein levels in adult liver, in which LPL protein was mainly localized onto sinusoidal epithelial cells and Kupffer cells. As a result, the LPL activity in postheparin plasma was substantially increased, and postprandial plasma triglyceride clearance was significantly enhanced, whereas plasma triglyceride levels were decreased. The dysregulated liver LPL expression and low plasma triglyceride levels in ZBTB20-deficient mice were normalized by inactivating hepatic LPL expression. ZBTB20 deficiency protected the mice against high-fat diet-induced hyperlipidemia without causing excessive triglyceride accumulation in the liver. Chromatin immunoprecipitation and gel-shift assay studies revealed that ZBTB20 binds to the LPL promoter in the liver. A luciferase reporter assay revealed that ZBTB20 inhibits the transcriptional activity of LPL promoter. The regulation of LPL expression by ZBTB20 is liver-specific under physiological conditions. CONCLUSIONS: Liver ZBTB20 serves as a key regulator of LPL expression and plasma triglyceride metabolism and could be a therapeutic target for hypertriglyceridemia.

The alleviating effect of sphingosine kinases 2 inhibitor K145 on nonalcoholic fatty liver.

Sphingosine kinase 2 (SphK2) inhibitors are developed for tumor therapy as considering its anti-tumor effect. Many studies also explored SphK2 modulated glucose and lipid homeostasis, which extended its potential function for metabolic diseases therapy. In this study, we discovered a significant reduction of hepatic lipid accumulation as well as recovery of liver function in ob/ob mice with intraperitoneal injection of K145. Also, db/db mice received K145 showed improvement of both NALFD and hyperglycemia. We furtherly analyzed the genes associated with lipid metabolism and found a remarkable decreased expression of lipogenic genes including FAS, ACC1 and SREBP1c whereas elevated mitochondrial fatty acid ß-oxidation (FAO) related genes expression including CPT1A, MCAD, LCAD, PPAR-α, UCP2. Consistent to in vivo study, in vitro study also confirmed the role of K145 in decreasing lipid accumulation in human HL7702 cells, while inhibiting FAS, ACC1 and SREBP1c mRNA expression. It indicated a possible mechanism of K145 induced improvement of hepatic lipid accumulation partly via inhibition of lipigenesis. Our study suggested a promising role of K145 in drug development for NAFLD and diabetes therapy.

Fructose and metabolic diseases: too much to be good.

ABSTRACT: Excessive consumption of fructose, the sweetest of all naturally occurring carbohydrates, has been linked to worldwide epidemics of metabolic diseases in humans, and it is considered an independent risk factor for cardiovascular diseases. We provide an overview about the features of fructose metabolism, as well as potential mechanisms by which excessive fructose intake is associated with the pathogenesis of metabolic diseases both in humans and rodents. To accomplish this aim, we focus on illuminating the cellular and molecular mechanisms of fructose metabolism as well as its signaling effects on metabolic and cardiovascular homeostasis in health and disease, highlighting the role of carbohydrate-responsive element-binding protein in regulating fructose metabolism.

Increased lipoxygenase and decreased cytochrome P450s metabolites correlated with the incidence of diabetic nephropathy: Potential role of eicosanoids from metabolomics in type 2 diabetic patients.

Diabetic nephropathy (DN) is the major cause of chronic kidney disease and end-stage renal disease. Previous studies have demonstrated that long-chain omega-3 polyunsaturated fatty acids (PUFAs) might have therapeutic potential in reducing proteinuria in DN. However, the local level of eicosanoids derived from PUFAs in the plasma of DN patients remains unclear. This work aims to study the eicosanoid profile difference in plasma of DN patients and type 2 diabetes (T2D) without DN. A total of 27 T2D patients with similar diabetic duration were recruited and divided into T2D+DN group and T2D+NDN (non-DN) group based on urinary albumin excretion (UAE) detection. Using LC-MS/MS-based metabolomics, DN patients showed increased level of lipoxygenase (LOX) metabolites (5-HETE and LTB4) and decreased levels of eicosanoids derived according to the cytochrome P450s (CYP450) metabolic pathway (5,6-DHET; 14,15-DHET and 9,10-diHOME). Receiver operating characteristics and logistic regression analysis revealed increased level LOX metabolites and decreased level of CYP450 metabolites were significantly correlated with the incidence of DN in T2D patients. LOX and CYP450 metabolites correlated with DN incidence in T2D patients, which might be treatment targets for DN in T2D patients.

Harmine alleviates atherogenesis by inhibiting disturbed flow-mediated endothelial activation via protein tyrosine phosphatase PTPN14 and YAP.

BACKGROUND AND PURPOSE: Disturbed flow induces endothelial dysfunction and contributes to uneven distribution of atherosclerotic plaque. Emerging evidence suggests that harmine, a natural constituent of extracts of Peganum harmala, has potent beneficial activities. Here, we investigated if harmine has an atheroprotective role under disturbed flow and the underlying mechanism. EXPERIMENTAL APPROACH: Mice of ApoE-/- , LDLR-/- , and endothelial cell (EC)-specific overexpression of yes-associated protein (YAP) in ApoE-/- background were fed with a Western diet and given harmine for 4 weeks. Atherosclerotic lesion size, cellular composition, and expression of inflammatory genes in the aortic roots were assessed. HUVECs were treated with oscillatory shear stress (OSS) and harmine and also used for proteomic analysis. KEY RESULTS: Harmine retarded atherogenesis in both ApoE-/- and LDLR-/- mice by inhibiting the endothelial inflammatory response. Mechanistically, harmine blocked OSS-induced YAP nuclear translocation and EC activation by reducing phosphorylation of YAP at Y357. Overexpression of endothelial YAP blunted the beneficial effects of harmine in mice. Proteomic study revealed that protein tyrosine phosphatase non-receptor type 14 (PTPN14) could bind to YAP. Moreover, harmine increased PTPN14 expression by stabilizing its protein level and inhibiting its degradation in proteasomes. PTPN14 knockdown blocked the effects of harmine on YAPY357 and EC activation. Finally, overexpression of PTPN14 mimicked the effects of harmine and ameliorated atherosclerosis, and knockdown of PTPN14 blunted the atheroprotective effects of harmine and accelerated atherosclerosis, in a partial ligation mouse model. CONCLUSION AND IMPLICATIONS: Harmine alleviated OSS-induced EC activation via a PTPN14/YAPY357 pathway and had a potent atheroprotective role.

c-Abl regulates YAPY357 phosphorylation to activate endothelial atherogenic responses to disturbed flow.

Local flow patterns determine the uneven distribution of atherosclerotic lesions. This research aims to elucidate the mechanism of regulation of nuclear translocation of Yes-associated protein (YAP) under oscillatory shear stress (OSS) in the atheroprone phenotype of endothelial cells (ECs). We report here that OSS led to tyrosine phosphorylation and strong, continuous nuclear translocation of YAP in ECs that is dependent on integrin α5ß1 activation. YAP overexpression in ECs blunted the anti-atheroprone effect of an integrin α5ß1-blocking peptide (ATN161) in Apoe-/- mice. Activation of integrin α5ß1 induced tyrosine, but not serine, phosphorylation of YAP in ECs. Blockage of integrin α5ß1 with ATN161 abolished the phosphorylation of YAP at Y357 induced by OSS. Mechanistic studies showed that c-Abl inhibitor attenuated the integrin α5ß1-induced YAP tyrosine phosphorylation. Furthermore, the phosphorylation of c-Abl and YAPY357 was significantly increased in ECs in atherosclerotic vessels of mice and in human plaques versus normal vessels. Finally, bosutinib, a tyrosine kinase inhibitor, markedly reduced the level of YAPY357 and the development of atherosclerosis in Apoe-/- mice. The c-Abl/YAPY357 pathway serves as a mechanism for the activation of integrin α5ß1 and the atherogenic phenotype of ECs in response to OSS, and provides a potential therapeutic strategy for atherogenesis.

Macrophage Raptor Deficiency-Induced Lysosome Dysfunction Exacerbates Nonalcoholic Steatohepatitis.

BACKGROUND & AIMS: Nonalcoholic steatohepatitis (NASH) is an increasingly prevalent nonalcoholic fatty liver disease, characterized by inflammatory cell infiltration and hepatocellular damage. Mammalian target of rapamycin complex 1 (mTORC1) has been investigated extensively in the context of cancer, including hepatocellular carcinoma. However, the role of mTORC1 in NASH remains largely unknown. METHODS: mTORC1 activity in macrophages in human mild and severe NASH liver was compared. Mice with macrophage-specific deletion of the regulatory-associated protein of mTOR (Raptor) subunit and littermate controls were fed a high-fructose, palmitate, and cholesterol diet for 24 weeks or a methionine- and choline-deficient diet for 4 weeks to develop NASH. RESULTS: We report that in human beings bearing NASH, macrophage mTORC1 activity was lower in livers experiencing severe vs mild NASH liver. Moreover, macrophage mTORC1 disruption exacerbated the inflammatory response in 2 diet-induced NASH mouse models. Mechanistically, in response to apoptotic hepatocytes (AHs), macrophage polarization toward a M2 anti-inflammatory phenotype was inhibited in Raptor-deficient macrophages. During the digestion of AHs, macrophage mTORC1 was activated and coupled with dynamin-related protein 1 to facilitate the latter's phosphorylation, leading to mitochondrial fission-mediated calcium release. Ionomycin or A23187, calcium ionophores, prevented Raptor deficiency-mediated failure of lysosome acidification and subsequent lipolysis. Blocking dynamin-related protein 1-dependent mitochondria fission impaired lysosome function, resulting in reduced production of anti-inflammatory factors such as interleukins 10 and 13. CONCLUSIONS: Persistent mTORC1 deficiency in macrophages contributes to the progression of NASH by causing lysosome dysfunction and subsequently attenuating anti-inflammatory M2-like response in macrophages during clearance of AHs.

Elevating ATP-binding cassette transporter G1 improves re-endothelialization function of endothelial progenitor cells via Lyn/Akt/eNOS in diabetic mice.

Endothelial progenitor cell (EPC) dysfunction contributes to diabetes-induced delay in endothelium repair after vessel injury, prominently associated with diabetic cardiovascular complications such as neointima formation. ATP-binding cassette transporter G1 (ABCG1) promotes cholesterol efflux to HDL, which may favorably affect EPC function. However, whether ABCG1 improves EPC function, especially in diabetes, remains unknown. Here we investigated the role of ABCG1 in EPCs by using Tie2-mediated ABCG1 transgenic (Tie2- ABCG1Tg) mice. Mice were injected with streptozotocin to induce diabetes mellitus. As compared with wild-type (WT) mice, in Tie2- ABCG1Tg mice, diabetes-impaired EPC migration and tube formation were reversed. In vitro gain-of-function and loss-of-function studies further revealed that ABCG1-overexpressing EPCs showed increased migration and tube formation and differentiation via the Lck/Yes-related novel protein tyrosine kinase /Akt/endothelial NO synthase pathway by enhancing cellular cholesterol efflux. Finally, type 1 and type 2 diabetic mouse models with arterial injury were intravenously injected with labeled EPCs from WT or Tie2- ABCG1Tg mice. Re-endothelialization in diabetic mice was improved to a greater extent by injection of ABCG1-overexpressing than WT EPCs. Our study demonstrated that ABCG1 in EPCs improved repair after vascular injury in diabetes by increasing EPC function such as migration, tube formation and differentiation, and subsequent re-endothelialization. ABCG1 might be a promising therapeutic target for diabetes-associated vascular diseases.-Shi, Y., Lv, X., Liu, Y., Li, B., Liu, M., Yan, M., Liu, Y., Li, Q., Zhang, X., He, S., Zhu, M., He, J., Zhu, Y., Zhu, Y., Ai, D. Elevating ATP-binding cassette transporter G1 improves re-endothelialization function of endothelial progenitor cells via Lyn/Akt/eNOS in diabetic mice.

Plasma metabolic profile reveals PGF2α protecting against non-proliferative diabetic retinopathy in patients with type 2 diabetes.

Diabetic retinopathy (DR) is the most frequent microvascular complications of diabetes and the leading cause of blindness in adults worldwide. Non-proliferative DR (NPDR) is the first stage of DR but currently has few recommended intervention. Eicosanoids play important roles in maintaining vessel homeostasis. However, the functions of eicosanoids in NPDR are still unknown. In this study, we investigated the eicosanoids profile difference in plasma between type 2 diabetes with NPDR or not. A total of 50 patients with type 2 diabetes were recruited and divided into non-DR (NDR) group and NPDR group based on fundus photographs. The eicosanoids profiles in plasma were determined by LC-MS/MS. Adhesion and transwell assay were used to detect the adhesion and migration effects of metabolites on primary bovine retinal pericyte cells (BRPC), respectively. Streptomycin (STZ)-induced diabetic mouse model was used to test the protective effects of selected metabolites according to retinal immunofluorescence staining and fluorescence confocal microscopy. Prostaglandin 2α (PGF2α) was decreased significantly in NPDR group compared to NDR group and negatively correlated with NPDR. In vitro, PGF2α was found to accelerate adhesion and migration by activating prostaglandin F receptor (FP receptor) and subsequent increasing RhoA activity in primary bovine retinal pericyte. Administration of PGF2α analogue diminished the damage on retinal capillary in an STZ-induced diabetic mouse model. Our results suggested that PGF2α may be a protective factor in the progression of NPDR in T2D patients. The protective mechanism of PGF2α was to increase pericyte mobility through FP receptor/RhoA pathway.

Yes-Associated Protein Promotes Angiogenesis via Signal Transducer and Activator of Transcription 3 in Endothelial Cells.

RATIONALE: Angiogenesis is a complex process regulating endothelial cell (EC) functions. Emerging lines of evidence support that YAP (Yes-associated protein) plays an important role in regulating the angiogenic activity of ECs. OBJECTIVE: The objective of this study was to specify the effect of EC YAP on angiogenesis and its underlying mechanisms. METHOD AND RESULTS: In ECs, vascular endothelial growth factor reduced YAP phosphorylation time and dose dependently and increased its nuclear accumulation. Using Tie2Cre-mediated YAP transgenic mice, we found that YAP promoted angiogenesis in the postnatal retina and tumor tissues. Mass spectrometry revealed signal transducer and activator of transcription 3 (STAT3) as a potential binding partner of YAP in ECs. Western blot and immunoprecipitation assays indicated that binding with YAP prolonged interleukin 6-induced STAT3 nuclear accumulation by blocking chromosomal maintenance 1-mediated STAT3 nuclear export without affecting its phosphorylation. Moreover, angiopoietin-2 expression induced by STAT3 was enhanced by YAP overexpression in ECs. Finally, a selective STAT3 inhibitor or angiopoietin-2 blockage partly attenuated retinal angiogenesis in Tie2Cre-mediated YAP transgenic mice. CONCLUSIONS: YAP binding sustained STAT3 in the nucleus to enhance the latter's transcriptional activity and promote angiogenesis via regulation of angiopoietin-2.

Spectrum evaluation-assisted eicosanoid metabolomics for global eicosanoid profiling in human vascular endothelial cells.

Eicosanoids are hundreds of metabolites derived from poly-unsaturated fatty acids (PUFAs), which regulate biological processes from multiple angles via a complex metabolic network. Targeted eicosanoid metabolomics is used to study the eicosanoid profile in biological samples but only for eicosanoids with available standards. To expand the coverage of eicosanoids detected, we identified the eicosanoids without available standards by estimation of the retention time and comparison of the MS/MS spectra with the reference ones which was collected in a database from literature. Scheduled multiple reaction monitoring- information dependent acquisition- enhanced product ion (sMRM-IDA-EPI) scan mode was applied in this method, which was called Spectrum Evaluation-assisted Eicosanoid Metabolomics (SEEM). By using this method, 243 eicosanoids (167 without standards) could be relatively quantified with precision over 90 percent. We applied the method to analyze the global profile of eicosanoids secreted by human umbilical vascular endothelial cells at the basal level and with n-3 PUFA treatment. 26 putative eicosanoids showed altered levels, despite no available standards. In general, n-3 PUFA treatment increased most of their own metabolites and decreased the epoxy-, hydroxyl- and keto- linoleic acid metabolites. The application of the SEEM method proved its potency of identification and quantification of eicosanoids without standards.

Metabolic profiling of murine plasma reveals eicosapentaenoic acid metabolites protecting against endothelial activation and atherosclerosis.

BACKGROUND AND PURPOSE: Atherosclerosis results from a maladaptive inflammatory response initiated by the intramural retention of LDL in susceptible areas of the arterial vasculature. The ω-3 polyunsaturated fatty acids (ω-3) have protective effects in atherosclerosis; however, their molecular mechanism is still largely unknown. The present study used a metabolomic approach to reveal the atheroprotective metabolites of ω-3 and investigate the underlying mechanisms. EXPERIMENTAL APPROACH: We evaluated the development of atherosclerosis in LDL receptor-deficient mice (LDLR-/- ) fed a Western-type diet (WTD) plus ω-3 and also LDLR-/- and fat-1 transgenic (LDLR-/- -fat-1tg ) mice fed a WTD. The profiles of ω-3 in the plasma were screened by LC-MS/MS using unbiased systematic metabolomics analysis. We also studied the effect of metabolites of eicosapentaenoic acid (EPA) on endothelial activation in vitro. KEY RESULTS: The ω-3 diet and fat-1 transgene decreased monocyte infiltration, inhibited the expression of pro-inflammatory genes and significantly attenuated atherosclerotic plaque formation and enhanced plaque stability in LDLR-/- mice. The content of 18-hydroxy-eicosapentaenoic acid (18-HEPE) and 17,18-epoxy-eicosatetraenoic acid (17,18-EEQ), from the cytochrome P450 pathway of EPA, was significantly higher in plasma from both ω-3-treated LDLR-/- and LDLR-/- -fat-1tg mice as compared with WTD-fed LDLR-/- mice. In vitro in endothelial cells, 18-HEPE or 17,18-EEQ decreased inflammatory gene expression induced by TNFα via NF-κB signalling and thereby inhibited monocyte adhesion to endothelial cells. CONCLUSIONS AND IMPLICATIONS: EPA protected against the development of atherosclerosis in atheroprone mice via the metabolites 18-HEPE and/or 17,18-EEQ, which reduced endothelial activation. These compounds may have therapeutic implications in atherosclerosis. LINKED ARTICLES: This article is part of a themed section on Spotlight on Small Molecules in Cardiovascular Diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.8/issuetoc.