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.

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.

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.

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.

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.

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.

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.

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.

Hyperhomocysteinemia activates the aryl hydrocarbon receptor/CD36 pathway to promote hepatic steatosis in mice.

UNLABELLED: Hyperhomocysteinemia (HHcy) is associated with liver diseases such as fatty liver and hepatic fibrosis; however, the underlying mechanism is still largely unknown. The current study aimed to explore the signaling pathway involved in HHcy-induced hepatic steatosis (HS). C57BL/6 mice were fed a high-methionine diet (HMD) for 4 and 8 weeks to establish the HHcy mouse model. Compared to a chow diet, the HMD induced hepatic steatosis and elevated hepatic expression of CD36, a fatty acid transport protein. The increased CD36 expression was associated with activation of the aryl hydrocarbon receptor (AHR). In primary cultured hepatocytes, high levels of homocysteine (Hcy) treatment up-regulated CD36 and increased subsequent lipid uptake; both were significantly attenuated by small interfering RNA (siRNA) knockdown of CD36 and AHR. Chromatin immunoprecipitation assay revealed that Hcy promoted binding of AHR to the CD36 promoter, and transient transfection assay demonstrated markedly increased activity of the AHR response element by Hcy, which was ligand dependent. Mass spectrometry revealed significantly increased hepatic content of lipoxin A4 (LXA4 ), a metabolite of arachidonic acid, in HMD-fed mice. Furthermore, overexpression of 15-oxoprostaglandin 13-reductase 1, a LXA4 inactivation enzyme, inhibited Hcy-induced AHR activation, lipid uptake, and lipid accumulation. Moreover, LXA4 -induced up-regulation of CD36 and lipid uptake was inhibited by AHR siRNA in vitro in hepatocytes. Finally, treatment with an AHR antagonist reversed HHcy-induced lipid accumulation by inhibiting the AHR-CD36 pathway in mice. CONCLUSION: HHcy activates the AHR-CD36 pathway by increasing hepatic LXA4 content, which results in hepatic steatosis. (Hepatology 2016;64:92-105).

Inhibition of soluble epoxide hydrolase alleviated atherosclerosis by reducing monocyte infiltration in Ldlr(-/-) mice.

RATIONALE: Circulating monocytes play pivotal roles in chronic inflammatory diseases. Epoxyeicosatrienoic acids (EETs), metabolites of arachidonic acid, are known to have anti-inflammatory effects and are hydrolyzed by soluble epoxide hydrolase (sEH). OBJECTIVE: We aimed to investigate the effect of sEH inhibition in atherogenesis. METHODS AND RESULTS: Mice with low-density lipoprotein receptor deficiency (Ldlr(-/-)) with or without sEH inhibitor, and Ldlr/sEH double-knockout (DK) mice were fed a Western-type diet (WTD) for 6weeks to induce arteriosclerosis. Both sEH inhibition and gene depletion decreased the WTD-induced hyperlipidemia, plaque area and macrophage infiltration in mice arterial wall. Ly6C(hi) infiltration of monocytes remained similar in blood, spleen and bone marrow of DK mice, but was decreased in aortic lesions. To further assess the role of sEH or EETs in monocyte/macrophage infiltration in atherogenesis, we transplanted DK bone marrow into Ldlr(-/-) recipients, and then fed mice the WTD. Aortic lesions and Ly6C(hi) monocyte infiltration were reduced in mice with transplanted bone marrow of DK mice without diminishing the cholesterol level. Furthermore, sEH inhibition or gene depletion increased the ratio of EETs/DHETs and diminished the expression of P-selectin glycoprotein ligand 1 (PSGL-1) in mice peripheral-blood mononuclear cells. Monocyte adhesion to P-selectin and to tumor necrosis factor α-activated endothelial cells was also diminished by sEH inhibition. CONCLUSION: sEH inhibition and gene depletion attenuated atherosclerosis in mice by decreasing the infiltration of monocytes into the artery wall. EET and PSGL-1 may play pivotal roles in monocyte/macrophage infiltration and atherogenesis.

Metabolomic analysis revealed the role of DNA methylation in the balance of arachidonic acid metabolism and endothelial activation.

Arachidonic acid (AA) metabolism plays an important role in vascular homeostasis. We reported that DNA hypomethylation of EPHX2 induced a pro-inflammatory response in vascular endothelial cells (ECs). However, the change in the whole AA metabolism by DNA methylation is still unknown. Using a metabolomic approach, we investigated the effect of DNA methylation on the balance of AA metabolism and the underlying mechanism. ECs were treated with a DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-AZA), and AA metabolic profiles were analyzed. Levels of prostaglandin D2 (PGD2) and thromboxane B2 (TXB2), metabolites in the cyclooxygenase (COX) pathway, were significantly increased by 5-AZA treatment in ECs resulting from the induction of PGD2 synthase (PTGDS) and thromboxane A synthase 1 (TBXAS1) expression by DNA hypomethylation. This phenomenon was also observed in liver and kidney cell lines, indicating a universal mechanism. Pathophysiologically, homocysteine, known to cause DNA demethylation, induced a similar pattern of the change of AA metabolism. Furthermore, 5-AZA activated ECs, as evidenced by the upregulation of adhesion molecules. Indomethacin, a COX inhibitor, reversed the effects of 5-AZA on the levels of PGD2 and TXB2, EC activation and monocyte adhesion. In vivo, the plasma levels of PGD2 and TXB2 and the expression of In vivo PTGDS and TBXAS1 as well as adhesion molecules were increased in the aorta of the mice injected with 5-AZA. In conclusion, using a metabolomic approach, our study uncovered that DNA demethylation increased AA metabolites PGD2 and TXB2 by upregulating the expression of the corresponding enzymes, which might contribute to the DNA hypomethylation-induced endothelial activation.

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.

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.

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.

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.

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.