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Nϵ-carboxymethyl-lysine (CML), an advanced glycation end product, is involved in vascular calcification (VC) in diabetic atherosclerosis. This study aimed to investigate the effects of CML on VC in diabetic atherosclerosis induced by vascular smooth muscle cell (VSMC)-derived foam cells. Human studies, animal studies and cell studies were performed. The human study results from 100 patients revealed a poor blood glucose and lipid status and more severe coronary lesions and stenosis in patients with coronary artery disease and diabetes mellitus. Intraperitoneal injection of streptozotocin combined with a high-fat diet was used to build a diabetic atherosclerosis model in ApoE-/- mice. The animal study results indicated that CML accelerated VC progression in diabetic atherosclerosis by accelerating the accumulation of VSMC-derived foam cells in ApoE-/- mice. The cell study results illustrated that CML induced VSMC-derived foam cells apoptosis and aggravated foam cells calcification. Consistent with this finding, calcium content and the expression levels of alkaline phosphatase, bone morphogenetic protein 2 and runt-related transcription factor 2 were significantly elevated in A7r5 cells treated with oxidation-low-density lipoprotein and CML. Thus, we concluded that CML promoted VSMC-derived foam cells calcification to aggravate VC in diabetic atherosclerosis, providing evidence for the contribution of foam cells to diabetic VC.

Smoking is highly associated with cardiovascular diseases. However, the effect of nicotine, a key ingredient in smoking products, on cardiomyocyte apoptosis remains controversial. The present study aims to clarify the role of nicotine on cardiomyocyte cell apoptosis and to investigate the underlying mechanism. In the present study, H9C2 cells were exposed to nicotine at various concentrations (0, 10 and 100 µM) for 48 h. Cell Counting Kit‑8 and TUNEL assays were performed to assess cell viability and apoptosis, respectively, and reverse transcription‑quantitative polymerase chain reaction and western blot analysis were used to investigate the mRNA and protein expression. PYR‑41, a ubiquitin E1 inhibitor, was employed to investigate whether the ubiquitin‑proteasome system was involved in the downregulation of Akt. An Akt1 overexpression plasmid was used to demonstrate the role of Akt in H9C2 cells apoptosis. Tetratricopeptide repeat domain 3 (TTC3) small interfering RNA (siRNA) was used to investigate the effect of TTC3 on Akt protein degradation. The results demonstrated that nicotine induced apoptosis in H9C2 cells compared with control cells (P

Adipose-derived stem cells (ADSCs) have the ability to migrate to injury sites and facilitate tissue repair by promoting angiogenesis. However, the therapeutic effect of ADSCs from patients with diabetes is impaired due to oxidative stress. Given that diabetes is a group of metabolic disorders and mitochondria are a major source of reactive oxygen species (ROS), it is possible that mitochondrial ROS plays an important role in the induction of diabetic ADSC (dADSC) dysfunction. ADSCs isolated from diabetic mice were treated with mitoTEMPO, a mitochondrial ROS scavenger, or TEMPO, a universal ROS scavenger, for three passages. The results showed that pretreatment with mitoTEMPO increased the proliferation, multidifferentiation potential, and the migration and proangiogenic capacities of dADSCs to levels similar to those of ADSCs from control mice, whereas pretreatment with TEMPO showed only minor effects. Mechanistically, mitoTEMPO pretreatment enhanced the mitochondrial antioxidant capacity of dADSCs, and knockdown of superoxide dismutase reduced the restored mitochondrial antioxidant capacity and attenuated the proangiogenic effects induced by mitoTEMPO pretreatment. In addition, mitoTEMPO pretreatment improved the survival of dADSCs in diabetic mice with critical limb ischemia, showing protective effects similar to those of control ADSCs. Pretreatment of dADSCs with mitoTEMPO decreased limb injury and improved angiogenesis in diabetic mice with critical limb ischemia. These findings suggested that short-term pretreatment of dADSCs with a mitochondrial ROS scavenger restored their normal functions, which may be an effective strategy for improving the therapeutic effects of ADSC-based therapies in patients with diabetes.

Objective Adiponectin (APN) circulates as high‐molecular weight (HMW), medium‐molecular weight (MMW), and low‐molecular weight (LMW) forms. Nonalcoholic fatty liver disease (NAFLD) is a common cause of chronic liver disease. Currently, the role of LMW, MMW, and HMW APN remains largely unclear in NAFLD. Methods We examined the variation of these forms and analyzed the related clinical characteristics in NAFLD. A total of 63 male NAFLD patients (mean age: 43.00 ± 6.10 years) and 70 healthy male subjects (mean age: 42.53 ± 7.98 years) were included in the study. Total APN and other clinical characteristics were measured. The changes in HMW, MMW, and LMW APN were determined in NAFLD patients and NAFLD patients on a high‐fat diet, and the association between the groups was further analyzed. Results Decreased levels of total APN and three APN isoforms were found in NAFLD. Significantly decreased levels of HMW (P

Branched chain amino acids (BCAAs) are associated with the progression of obesity-related metabolic disorders, including type 2 diabetes and nonalcoholic fatty liver disease. However, whether BCAAs disrupt the homeostasis of hepatic glucose and lipid metabolism remains unknown. In this study, we observed that BCAAs supplementation significantly reduced high-fat (HF) diet–induced hepatic lipid accumulation while increasing the plasma lipid levels and promoting muscular and renal lipid accumulation. Further studies demonstrated that BCAAs supplementation significantly increased hepatic gluconeogenesis and suppressed hepatic lipogenesis in HF diet-induced obese (DIO) mice. These phenotypes resulted from severe attenuation of Akt2 signaling via mTORC1- and mTORC2-dependent pathways. BCAAs/branched-chain α-keto acids (BCKAs) chronically suppressed Akt2 activation through mTORC1 and mTORC2 signaling and promoted Akt2 ubiquitin-proteasome–dependent degradation through the mTORC2 pathway. Moreover, the E3 ligase Mul1 played an essential role in BCAAs/BCKAs-mTORC2-induced Akt2 ubiquitin-dependent degradation. We also demonstrated that BCAAs inhibited hepatic lipogenesis by blocking Akt2/SREBP1/INSIG2a signaling and increased hepatic glycogenesis by regulating Akt2/Foxo1 signaling. Collectively, these data demonstrate that in DIO mice, BCAAs supplementation resulted in serious hepatic metabolic disorder and severe liver insulin resistance: insulin failed to not only suppress gluconeogenesis but also activate lipogenesis. Intervening BCAA metabolism is a potential therapeutic target for severe insulin-resistant disease.

Background Contrast-induced nephropathy (CIN) is one of major and serious complications in patients undergoing percutaneous coronary intervention (PCI). It is unknown whether increased urinary adiponectin (UAPN), a sensitive marker for early renal function impairment, is associated with an increased risk of CIN. Therefore, we prospectively investigate the association of UAPN with CIN. Methods We prospectively enrolled 208 patients who were undergoing elective PCI. The baseline UAPN was assessed prior to PCI. The ROC analysis was used to evaluate the predictive value of UAPN for CIN. Multivariate logistic regression analysis was performed to analyze the independent risk factors for CIN. Results Of 208 patients, CIN occurred in 19 patients (9.13%), and 6 of them (2.88%) required dialysis. Patients with CIN had a higher UAPN level than those without CIN (17.15 ± 12.36 vs. 10.29 ± 3.04 ng/ml, P 124 μmol/L (OR, 4.210; 95% CI, 1.297–13.669; P

Diabetic mellitus (DM) portends poor prognosis concerning pressure overloaded heart disease. Branched-chain amino acids (BCAAs), elements of essential amino acids, have been found altered in its catabolism in diabetes decades ago. However, the relationship between BCAAs and DM induced deterioration of pressure overloaded heart disease remains controversial. This study is aimed to investigate the particular effect of BCKA, a metabolite of BCAA, on myocardial injury induced by pressure overloaded. Primary cardiomyocytes were incubated with or without BCKA and followed by treatment with isoproterenol (ISO); then cell viability was detected by CCK8 and apoptosis was examined by TUNNEL stain and caspase-3 activity analysis. Compared to non-BCKA incubated group, BCKA incubation decreased cell survival and increased apoptosis concentration dependently. Furthermore, Western blot assay showed that mTORC2-Akt pathway was significantly inactivated by BCKA incubation. Moreover, overexpression of rictor, a vital component of mTORC2, significantly abolished the adverse effects of BCKA on apoptosis susceptibility of cardiomyocytes. These results indicate that BCKA contribute to vulnerability of cardiomyocytes in stimulated stress via inactivation of mTORC2-Akt pathway.

Aim: This study was to investigate the change of high-molecula-weight (HMW) adiponectin (APN) isoform, the association between type 2 diabetes mellitus (T2DM) and HMW APN isoform, the variation of Disulfide-bond A oxidoreductase-like protein (DsbA-L), the effect of HMW APN isoform on AMP-dependent protein kinase (AMPK) and endothelial nitric oxide synthase (eNOS) in Chinese T2DM. Method: 169 patients aged at (48.7±9.4) years and 107 healthy control subjects aged at (42.6±7.8) years took part in this study. Anthropometric measures of the characters were assayed and different APN isoforms, DsbA-L, AMPK and eNOS levels were determined. Results: Ln(sRAGE) and Ln(Adiponectin) were significantly lower and significantly higher for the other characteristics in T2DM. Ln(Adiponectin) was negatively and significantly correlated with WHR, Ln(triglycerides), fasting plasma glucose, HbA1c (%) in control subjects and T2DM patients. Plasma and adipose tissue total APN and HMW APN were significantly reduced in newly diagnosed T2DM patients. DsbA-L was markedly down-regulated in diabetic adipose tissue. HMW APN caused significant decreases in AMPK and eNOS phosphorylation levels of human umbilical vein endothelial cells (HUVECs). Conclusions: Our results demonstrated that total APN levels was closely related to the risk of T2DM and HMW APN reduction was involved in the diabetic vascular AMPK/eNOS signal pathway. The findings will provide insight into novel therapeutic approaches for reducing the elevated cardiovascular risk associated with T2DM.

Background Biochemical marker has revolutionized the approach to the diagnosis of heart failure. However, it remains difficult to assess stability of the patient. As such, novel means of stratifying disease severity are needed. C1q/TNF-Related Protein 3 (CTRP3) and C1q/TNF-Related Protein 9 (CTRP9) are novel adipokines that contribute to energy homeostasis with additional anti-inflammatory and anti-ischemic properties. The aim of our study is to evaluate concentrations of CTRP3 and CTRP9 in patients with HFrEF (heart failure with reduced ejection fraction) and whether associated with mortality. Methods Clinical data and plasma were obtained from 176 healthy controls and 168 patients with HFrEF. CTRP3 and CTRP9 levels were evaluated by enzyme-linked immunosorbent assay. Results Both CTRP3 and CTRP9 concentrations were significantly decreased in the HFrEF group compared to the control group (p

Vascular complications are the major causes of death in patients with diabetes, and endothelial dysfunction is the earliest event in vascular complications of diabetes. It has been reported that plasma irisin level is significantly reduced in patients with type 2 diabetic patients. The present study aimed to investigate whether irisin improved endothelial function in type 2 diabetes as well as the underlying mechanisms. The type 2 diabetes model was established by feeding C57BL/6 mice with high-fat diet. The type 2 diabetic mice exhibited reduced serum irisin level and impaired endothelial function. Irisin treatment (0.5 mg/kg/d) for two weeks improved vascular function based on the evaluation of endothelium-dependent vasorelaxation and p-VASP levels. To investigate the direct endothelial protective effects of irisin, diabetic aortic segments were incubated with irisin (1 μg/ml) ex vivo. Exposure to irisin improved endothelium-dependent vasorelaxation of diabetic aortas. Mechanically, the diabetic aortic segments exhibited increased oxidative/nitrative stresses. Irisin reduced the diabetes-induced oxidative/nitrative stresses evidenced by reducing overproduction of superoxide and peroxynitrite, and down-regulation of iNOS and gp91(phox). To further investigate the protective effects of irisin on endothelial cells and the underlying mechanisms, human umbilical vein endothelial cells (HUVECs) cultured in high-glucose/high-fat (HG/HF) medium were pre-incubated with irisin. Irisin (1 μg/ml) reduced the oxidative/nitrative stresses and apoptosis induced by HG/HF in HUVECs probably via inhibiting activation of PKC-β/NADPH oxidase and NF-κB/iNOS pathways. Taken together, irisin alleviates endothelial dysfunction in type 2 diabetes partially via reducing oxidative/nitrative stresses through inhibiting signaling pathways implicating PKC-β/NADPH oxidase and NF-κB/iNOS, suggesting that irisin may be a promising molecule for the treatment of vascular complications of diabetes.

Diabetic patients are sensitive to myocardial ischemia-reperfusion (MI/R) injury. During diabetes, branched-chain amino acid (BCAA) catabolism is defective and mitochondrial phosphatase 2C (PP2Cm) expression is reduced. This study aims to elucidate the relationship between PP2Cm downregulation and BCAA catabolism defect in diabetic mice against MI/R injury. PP2Cm was significantly downregulated in hearts of diabetic mice. The cardiac function was improved and the myocardial infarct size and apoptosis were decreased in diabetic mice overexpressing PP2Cm after MI/R. In diabetic mice, the cardiac BCAA and its metabolites branched-chain keto-acids (BCKA) levels, and p-BCKDE1α (E1 subunit of BCKA dehydrogenase)/BCKDE1α ratio were increased while the BCKD activity was decreased. Treatment of diabetic mice subjected to MI/R injury with BT2, a BCKD kinase (BDK) inhibitor, alleviated the BCAA catabolism defect, and improved the cardiac function alongside reduced apoptosis. PP2Cm overexpression alleviated the BCAA catabolism defect and MI/R injury. Similarly, MnTBAP ameliorated the oxidative stress and MI/R injury. BCKA treatment of H9C2 cells under simulated ischemia/reperfusion (SI/R) injury significantly decreased cell viability and increased LDH release and apoptosis. These effects were alleviated by BT2 and MnTBAP treatments. These results suggested that PP2Cm directly mediates the BCAA catabolism defect and oxidative stress observed after MI/R in diabetes. Overexpression of PP2Cm alleviates MI/R injury by reducing the catabolism of BCAA and oxidative stress.

The branched-chain amino acids (BCAA) accumulated in type 2 diabetes are independent contributors to insulin resistance. The activity of branched-chain α-keto acid dehydrogenase (BCKD) complex, rate-limiting enzyme in BCAA catabolism, is reduced in diabetic states, which contributes to elevated BCAA concentrations. However, the mechanisms underlying decreased BCKD activity remain poorly understood. Here, we demonstrate that mitochondrial phosphatase 2C (PP2Cm), a newly identified BCKD phosphatase that increases BCKD activity, was significantly downregulated in ob/ob and type 2 diabetic mice. Interestingly, in adiponectin (APN) knockout (APN−/−) mice fed with a high-fat diet (HD), PP2Cm expression and BCKD activity were significantly decreased, whereas BCKD kinase (BDK), which inhibits BCKD activity, was markedly increased. Concurrently, plasma BCAA and branched-chain α-keto acids (BCKA) were significantly elevated. APN treatment markedly reverted PP2Cm, BDK, BCKD activity, and BCAA and BCKA levels in HD-fed APN−/− and diabetic animals. Additionally, increased BCKD activity caused by APN administration was partially but significantly inhibited in PP2Cm knockout mice. Finally, APN-mediated upregulation of PP2Cm expression and BCKD activity were abolished when AMPK was inhibited. Collectively, we have provided the first direct evidence that APN is a novel regulator of PP2Cm and systematic BCAA levels, suggesting that targeting APN may be a pharmacological approach to ameliorating BCAA catabolism in the diabetic state.

Cardiac metabolic remodeling is a central event during heart failure (HF) development following myocardial infarction (MI). It is well known that myocardial glucose and fatty acid dysmetabolism contribute to post-MI cardiac dysfunction and remodeling. However, the role of amino acid metabolism in post-MI HF remains elusive. Branched chain amino acids (BCAAs) are an important group of essential amino acids and function as crucial nutrient signaling in mammalian animals. The present study aimed to determine the role of cardiac BCAA metabolism in post-MI HF progression. Utilizing coronary artery ligation-induced murine MI models, we found that myocardial BCAA catabolism was significantly impaired in response to permanent MI, therefore leading to an obvious elevation of myocardial BCAA abundance. In MI-operated mice, oral BCAA administration further increased cardiac BCAA levels, activated the mammalian target of rapamycin (mTOR) signaling, and exacerbated cardiac dysfunction and remodeling. These data demonstrate that BCAAs act as a direct contributor to post-MI cardiac pathologies. Furthermore, these BCAA-mediated deleterious effects were improved by rapamycin cotreatment, revealing an indispensable role of mTOR in BCAA-mediated adverse effects on cardiac function/structure post-MI. Of note, pharmacological inhibition of branched chain ketoacid dehydrogenase kinase (BDK), a negative regulator of myocardial BCAA catabolism, significantly improved cardiac BCAA catabolic disorders, reduced myocardial BCAA levels, and ameliorated post-MI cardiac dysfunction and remodeling. In conclusion, our data provide the evidence that impaired cardiac BCAA catabolism directly contributes to post-MI cardiac dysfunction and remodeling. Moreover, improving cardiac BCAA catabolic defects may be a promising therapeutic strategy against post-MI HF.

The advanced glycation end products (AGEs) are associated with increased cardiac endothelial injury. However, no causative link has been established between increased AGEs and enhanced endothelial injury after ischemia/reperfusion. More importantly, the molecular mechanisms by which AGEs may increase endothelial injury remain unknown. Adult rat cardiac microvascular endothelial cells (CMECs) were isolated and incubated with AGE-modified bovine serum albumin (BSA) or BSA. After AGE-BSA or BSA preculture, CMECs were subjected to simulated ischemia (SI)/reperfusion (R). AGE-BSA increased SI/R injury as evidenced by enhanced lactate dehydrogenase release and caspase-3 activity. Moreover, AGE-BSA significantly increased SI/R-induced oxidative/nitrative stress in CMECs (as measured by increased inducible nitric oxide synthase expression, total nitric oxide production, superoxide generation, and peroxynitrite formation) and increased SI/R-induced nitrative inactivation of thioredoxin-1 (Trx-1), an essential cytoprotective molecule. Supplementation of EUK134 (peroxynitrite decomposition catalyst), human Trx-1, or soluble receptor of advanced end product (sRAGE) (a RAGE decoy) in AGE-BSA precultured cells attenuated SI/R-induced oxidative/nitrative stress, reduced SI/R-induced Trx-1 nitration, preserved Trx-1 activity, and reduced SI/R injury. Our results demonstrated that AGEs may increase SI/R-induced endothelial injury by increasing oxidative/nitrative injury and subsequent nitrative inactivation of Trx-1. Interventions blocking RAGE signaling or restoring Trx activity may be novel therapies to mitigate endothelial ischemia/reperfusion injury in the diabetic population.

Diets that are high in dietary fiber are reported to have substantial health benefits. We sought to compare the metabolic effects of 3 types of dietary fibers -- sugarcane fiber (SCF), psyllium (PSY), and cellulose (CEL) -- on body weight, carbohydrate metabolism, and stomach ghrelin gene expression in a high-fat diet-fed mouse model. Thirty-six male mice (C57BL/6) were randomly divided into 4 groups that consumed high-fat diet alone (HFD) or high-fat diet containing 10% SCF, PSY, and CEL, respectively. After baseline measurements were assessed for body weight, plasma insulin, glucose, leptin, and glucagon-like peptide 1 (GLP-1), animals were treated for 12 weeks. Parameters were reevaluated at the end of study. Whereas there was no difference at the baseline, body weight gains in the PSY and SCF groups were significantly lower than in the CEL group at the end of study. No difference in body weight was observed between the PSY and SCF animals. Body composition analysis demonstrated that fat mass in the SCF group was considerably lower than in the CEL and HFD groups. In addition, fasting plasma glucose and insulin and areas under the curve of intraperitoneal glucose tolerance test were also significantly lower in the SCF and PSY groups than in the CEL and HFD groups. Moreover, fasting plasma concentrations of leptin were significantly lower and GLP-1 level was 2-fold higher in the SCF and PSY mice than in the HFD and CEL mice. Ghrelin messenger RNA levels of stomach in the SCF group were significantly lower than in the CEL and HFD groups as well. These results suggest differences in response to dietary fiber intake in this animal model because high-fat diets incorporating dietary fibers such as SCF and PSY appeared to attenuate weight gain, enhance insulin sensitivity, and modulate leptin and GLP-1 secretion and gastric ghrelin gene expression.

It has been reported that irisin regulated exercise-mediated adipocyte browning; however, the systematical effects of irisin on the metabolism of glucose and lipid in diabetes are largely unknown. In the present study, we investigated the role and underlying mechanism of irisin in glucose utilization and lipid metabolism in diabetic mice.A mouse model of diabetes was established by feeding C57BL/6 mice with high-fat diet. The diabetic mice were then treated with irisin. To mimic type 2 diabetes in vitro, myocytes and hepatocytes were cultured in a medium of high glucose and high fat. Glucose uptake, fatty acid oxidation and the expression of related protein were evaluated.Irisin improved glucose tolerance and glucose uptake as evidenced by increased (18)F-FDG accumulation and GLUT4 translocation in diabetic skeletal muscle. Irisin also increased glucose uptake in myocytes cultured in high glucose/high fatty acid medium. In contrast, irisin reduced the expression of PEPCK and G6Pase, which are involved in gluconeogenesis, in diabetic liver. Consistently, irisin reduced fat weight and serum total cholesterol and triglyceride levels in diabetic mice, but increased acetyl coenzyme A carboxylase-β phosphorylation in muscle tissue and uncoupling protein 1 expression in fat tissue. In addition, irisin increased the oxidation of fatty acid in myocytes. Knockdown of the adenosine monophosphate (AMP)-activated protein kinase (AMPK) attenuated the effects of irisin on glucose uptake and fatty acid β-oxidation in myocytes. Similarly, inhibition of AMPK by a specific inhibitor reduced the effects of irisin on PEPCK and G6Pase expression in hepatocytes.Our results suggest that irisin has an essential role in glucose utilization and lipid metabolism, and irisin is a promising pharmacological target for the treatment of diabetes and its complications.

NLRP3 inflammasome is necessary for initiating acute sterile inflammation. Recent studies have demonstrated that NLRP3 inflammasome is up-regulated and mediates myocardial ischemia/reperfusion (MI/R) injury. However, the signaling pathways that lead to the activation of NLRP3 inflammasome by MI/R injury have not been fully elucidated. C57BL/6J mice were subjected to 30 min ischemia and 3 or 24 h reperfusion. The ischemic heart exhibited enhanced inflammasome activation as evidenced by increased NLRP3 expression and caspase-1 activity and increased IL-1β and IL-18 production. Intramyocardial NLRP3 siRNA injection or an intraperitoneal injection of BAY 11-7028, an inflammasome inhibitor, attenuated macrophage and neutrophil infiltration and decreased MI/R injury, as measured by cardiomyocyte apoptosis and infarct size. The ischemic heart also exhibited enhanced interaction between Txnip and NLRP3, which has been shown to be a mechanism for activating NLRP3. Intramyocardial Txnip siRNA injection also decreased infarct size and NLRP3 activation. In vitro experiments revealed that NLRP3 was expressed in cardiac microvascular endothelial cells (CMECs), but was hardly expressed in cardiomyocytes. Simulated ischemia/reperfusion (SI/R) stimulated NLRP3 inflammasome activation in CMECs, but not in cardiomyocytes. Moreover, CMECs subjected to SI/R injury increased interactions between Txnip and NLRP3. Txnip siRNA diminished NLRP3 inflammasome activation and SI/R-induced injury, as measured by LDH release and caspase-3 activity in CMECs. ROS scavenger dissociated TXNIP from NLRP3 and inhibited the activation of NLRP3 inflammasome in the CMECs. For the first time, we demonstrated that TXNIP-mediated NLRP3 inflammasome activation in CMECs was a novel mechanism of MI/R injury. Interventions that block Txnip/NLRP3 signaling to inhibit the activation of NLRP3 inflammasomes may be novel therapies for mitigating MI/R injury.

Impaired mitochondrial biogenesis causes skeletal muscle damage in diabetes. However, whether and how mitochondrial biogenesis is impaired in the diabetic heart remains largely unknown. Whether adiponectin (APN), a potent cardioprotective molecule, regulates cardiac mitochondrial function has also not been previously investigated. In this study, electron microscopy revealed significant mitochondrial disorders in ob/ob cardiomyocytes, including mitochondrial swelling and cristae disorientation and breakage. Moreover, mitochondrial biogenesis of ob/ob cardiomyocytes is significantly impaired, as evidenced by reduced Ppargc-1a/Nrf-1/Tfam mRNA levels, mitochondrial DNA content, ATP content, citrate synthase activity, complexes I/III/V activity, AMPK phosphorylation, and increased PGC-1α acetylation. Since APN is an upstream activator of AMPK and APN plasma levels are significantly reduced in ob/ob mice, we further tested the hypothesis that reduced APN in ob/ob mice is causatively related to mitochondrial biogenesis impairment. One week of APN treatment of ob/ob mice activated AMPK, reduced PGC-1α acetylation, increased mitochondrial biogenesis, and attenuated mitochondrial disorders. In contrast, knocking out APN inhibited AMPK-PGC-1α signaling and impaired both mitochondrial biogenesis and function. The ob/ob mice exhibited lower survival rates and exacerbated myocardial injury after MI, when compared to controls. APN supplementation improved mitochondrial biogenesis and attenuated MI injury, an effect that was almost completely abrogated by the AMPK inhibitor compound C. In high glucose/high fat treated neonatal rat ventricular myocytes, siRNA-mediated knockdown of PGC-1α blocked gAd-enhanced mitochondrial biogenesis and function and attenuated protection against hypoxia/reoxygenation injury. In conclusion, hypoadiponectinemia impaired AMPK-PGC-1α signaling, resulting in dysfunctional mitochondrial biogenesis that constitutes a novel mechanism for rendering diabetic hearts more vulnerable to enhanced MI injury.

Contrast-induced nephropathy (CIN) is one of major complications in patients undergoing PCI. We prospectively examined the association of urinary adiponectin (UAPN), a sensitive marker for early renal function impairment, with CIN. We enrolled 208 patients without severe hepatic and renal function

The receptor for advanced glycation end products (RAGE) and thioredoxin (Trx) play opposing roles in diabetic myocardial ischemia-reperfusion (MI/R) injury. We recently demonstrated nitrative modification of Trx leads to its inactivation and loss of cardioprotection. The present study is to determine the relationship between augmented RAGE expression and diminished Trx activity pertaining to exacerbated MI/R injury in the diabetic heart. The diabetic state was induced in mice by multiple intraperitoneal low-dose streptozotocin injections. RAGE small-interfering RNA (siRNA) or soluble RAGE (sRAGE, a RAGE decoy) was via intramyocardial and intraperitoneal injection before MI/R, respectively. Mice were subjected to 30 min of myocardial infarction followed by 3 or 24 h of reperfusion. At 10 min before reperfusion, diabetic mice were randomized to receive EUK134 (peroxynitrite scavenger), recombinant hTrx-1, nitrated Trx-1, apocynin (a NADPH oxidase inhibitor), or 1400W [an inducible nitric oxide synthase (iNOS) inhibitor] administration. The diabetic heart manifested increased RAGE expression and Nε-(carboxymethyl)lysine (CML, major advanced glycation end product subtype) content, reduced Trx-1 activity, and increased Trx nitration after MI/R. RAGE siRNA or administration of sRAGE in diabetic mice decreased MI/R-induced iNOS and gp91phox expression, reduced Trx nitration, preserved Trx activity, and decreased infarct size. Apocynin or 1400W significantly decreased nitrotyrosine production and restored Trx activity. Conversely, administration of either EUK134 or reduced hTrx, but not nitrated hTrx, attenuated MI/R-induced superoxide production, RAGE expression, and CML content and decreased cardiomyocyte apoptosis in diabetic mice. Collectively, we demonstrate that RAGE modulates the MI/R injury in a Trx nitrative inactivation fashion. Conversely, nitrative modification of Trx blocked its inhibitory effect upon RAGE expression in the diabetic heart. This is the first direct evidence demonstrating the alternative cross talk between RAGE overexpression and nitrative Trx inactivation, suggesting that interventions interfering with their interaction may be novel means of mitigating diabetic MI/R injury.

Type 1 diabetes (T1DM) portends poor prognosis concerning ischemic heart disease. Adiponectin (APN), an adipocytokine possessing insulin sensitizing and metabolic regulatory effects, has been recognized as a potent cardioprotective molecule. However, the relationship between APN and T1DM remains controversial and the role of cardiac-derived APN in T1DM is unclear. This study is aimed to investigate the dynamic change of both plasma and cardiac-derived APN expressions in T1DM, and the particular role of cardiac-derived APN in T1DM against myocardial ischemia/reperfusion (MI/R) injury. T1DM was established via intraperitoneal injection of streptozocin and followed by twice-daily subcutaneous injection of insulin or vehicle for 14 days. Non-diabetic mice of wild type and APN knockout were subjected to insulin or vehicle injection. MI/R was induced in Langendorff-perfused hearts. Compared to non-diabetic mice, plasma APN levels of diabetic mice significantly increased at 7 days, and slightly decreased at 14 days, while cardiac-derived APN levels gradually decreased over time. The MI/R injury measured as infarct size and cardiomyocyte apoptosis nearly doubled in diabetic mice. 14 days of insulin treatment increased both plasma and cardiac-derived APN levels in diabetic mice and attenuated myocardial injury via increasing AMPK phosphorylation in T1DM, which was partly reversed by Compound C (an AMPK inhibitor). Moreover, APN deficiency aggravated MI/R injury and partly abolished the protective effect of insulin treatment against MI/R injury, which was associated with decreased AMPK phosphorylation. The results suggest that cardiac-derived APN stimulated by long-term insulin treatment in T1DM exerts cardioprotection against MI/R injury via myocardial AMPK activation.

Recent clinical observations have indicated that nonlethal mechanical trauma significantly increases myocardial infarction risk even in the presence of completely normal coronary arteries. We investigated the molecular mechanisms responsible for exacerbation of ischemic myocardial injury after nonlethal mechanical trauma with a special focus on the role of tumor necrosis factor α and its potential downstream effector adiponectin, a novel adipokine with anti-inflammatory and cardioprotective properties.Laboratory study.University research unit.Male adult adiponectin knockout mice and wild-type mice.The animals were subjected to nonlethal mechanical trauma using the Noble-Collip drum (40 rpm ± 5 mins) followed by myocardial ischemia/reperfusion injury 7 days posttrauma. We also investigated the effects of neutralizing tumor necrosis factor α with etanercept and exogenous adiponectin supplementation on ischemic myocardial injury after trauma.Trauma significantly sensitized myocardium to ischemia/reperfusion injury as evidenced by increased apoptosis, enlarged infarct size, and decreased cardiac function. Plasma adiponectin concentrations were reduced after traumatic injury (the nadir occurring 3 days posttrauma), an effect abrogated by etanercept-mediated tumor necrosis factor α blockade. The downregulation of adiponectin was accompanied by increased myocardial superoxide and nitric oxide generation and peroxynitrite formation. Both etanercept and exogenous adiponectin supplementation (on day 3 posttrauma or 10 mins before reperfusion on day 7 posttrauma) markedly inhibited oxidative/nitrative stress and ischemia/reperfusion injury in posttraumatic ischemic/reperfused hearts of wild-type mice, whereas only adiponectin supplementation (but not tumor necrosis factor α inhibition) substantially attenuated posttraumatic ischemia/reperfusion injury in adiponectin knockout mice.Tumor necrosis factor α-induced downregulation of adiponectin and the resultant enhanced oxidative/nitrative stress are involved in exacerbated posttraumatic ischemic myocardial injury. Therapeutic approaches blocking tumor necrosis factor α production or restoring adiponectin might have prophylactic value against secondary myocardial ischemic injury after a primary nonlethal mechanical trauma.

A series of polymer-based Polydimethylsiloxanes (PDMS) ball valves with different opening pressures have been developed for biomedical applications. By tuning different weight ratios of the two components (the base and the curing agent) of PDMS, the valves will have different opening pressures because of different stiffness of the PDMS materials. The curing conditions and mechanical properties of the PDMS material with different ratios have been thoroughly studied. The compressive Young's modulus of the material can be tuned from 310 KPa to 2.0 MPa. Such kind valves can be fabricated by injection molding, one of the cheapest fabrication techniques. Also such kind valve has no dead volume and easily to be integrated into micro-total analysis systems (μTAS).

Objectives Thioredoxin-1 (Trx) is an important antioxidant and antiapoptotic molecule and its activity can be altered by the modification of different amino acid sites. The oxidative stress stimulates apoptosis signal-regulating kinase 1 (ASK-1) signaling and thus induces apoptosis, which was inhibited by Trx treatment. This inhibition could be attenuated by nitrative inactivation of Trx induced by the oxidative stress. However, the underlying mechanism remains largely unknown. Therefore, the present study aims to examine whether Y49 in Trx is the nitrative modification site and further investigate whether the single mutation of Trx at site Y49 (Trx Y49F) resists nitrative inactivation and strengthens the Trx9s inhibition on ASK-1 mediated apoptosis. Methods and Results In contrast to Trx, in vitro exposure of Trx Y49F to SIN-1 (a protein nitration reagent) did not result in protein nitration. The interactions of Trx/Trx Y49F and ASK-1 were studied by cotransfection of Trx/Trx Y49F and ASK-1 plasmids into HEK293A cells. By treating cells with H 2 O 2 , Trx-ASK-1 complex formation was reduced and activity of ASK-1 and p38 MAPK increased. In contrast, mutation of Trx at site Y49 prevented Trx nitration, increased Trx activity, restored Trx-ASK-1 interaction, reduced ASK-1 and P38 MAPK activity, and attenuated caspase-3 activation (p 2 O 2 . Further animal experiments confirmed that compared with Trx, Trx Y49F treatment is more effective in protecting MI/R injury by enhancing Trx-ASK-1 interaction, inhibiting p38 MAPK signaling pathway, and subsequently decreasing post-ischemic cardiomyocyte apoptosis (p Conclusions Our results demonstrated for the first time that the oxidative stress results in nitration of Trx at site Y49. Nitrative inactivation of Trx can be prevented by Y49F mutation, which strengthens the Trx9s inhibition of ASK-1-mediated pathological apoptosis. This finding may lead to a novel therapeutic strategy to attenuate MI/R injury in patients.

Zhong Q Wang,1,2 Yongmei Yu,1,2 Xian H Zhang,1,2 Z Elizabeth Floyd,3 Anik Boudreau,2 Kun Lian,4 William T Cefalu1,21Nutrition and Diabetes Research Laboratory, 2Botanical Research Center, 3Ubiquitin Laboratory, Pennington Biomedical Research Center, Louisiana State University System, Baton Rouge, LA, USA; 4The Center for Energy and Environmental Studies, Southern University, Baton Rouge, LA, USAAim: To compare the effects of dietary fibers on hepatic cellular signaling in mice.Methods: Mice were randomly divided into four groups (n = 9/group): high-fat diet (HFD) control, cellulose, psyllium, and sugarcane fiber (SCF) groups. All mice were fed a HFD with or without 10% dietary fiber (w/w) for 12 weeks. Body weight, food intake, fasting glucose, and fasting insulin levels were measured. At the end of the study, hepatic fibroblast growth factor (FGF) 21, AMP-activated protein kinase (AMPK) and insulin signaling protein content were determined.Results: Hepatic FGF21 content was significantly lowered, but βKlotho, fibroblast growth factor receptor 1, fibroblast growth factor receptor 3, and peroxisome proliferator-activated receptor alpha proteins were significantly increased in the SCF group compared with those in the HFD group (P < 0.01). SCF supplementation also significantly enhanced insulin and AMPK signaling, as well as decreased hepatic triglyceride and cholesterol in comparison with the HFD mice. The study has shown that dietary fiber, especially SCF, significantly attenuates lipid accumulation in the liver by enhancing hepatic FGF21, insulin, and AMPK signaling in mice fed a HFD.Conclusion: This study suggests that the modulation of gastrointestinal factors by dietary fibers may play a key role in both enhancing hepatic multiple cellular signaling and reducing lipid accumulation.Keywords: dietary fiber, FGF21, insulin signaling, AMPK, GLP-1, PI 3K

Objective Adiponectin, circulates as trimer, hexamer and high molecular weight form (HMW) in blood. Both adiponectin complexes concentration and their bioactivities are decreased in pathological state. Moreover, DsbA-L, a key regulator for adiponectin biosynthesis, is down-regulated in obese humans. However, the alterations of adiponectin multimers distribution, bioactivities and DsbA-L level in newly diagnosed Chinese type 2 diabetes (T2DM) patients are unknown. The objective of present study was to compare plasma adiponectin isoform status along with their bioactivities and DsbA-L concentration in T2DM with control subjects. Methods Plasma total adiponectin was measured by ELISA. Adiponectin multimers were analysed by western blot followed SDS-PAGE under nonheating nonreducing conditions. Plasma adiponectin multimers were isolated by chromotography techniques from plasma in ten T2DM and ten healthy control subjects respectively. Adiponectin bioactivity was detected by incubating HUVEC with isolated adiponectin isoforms and determining the phosphorylation level of AMPK and eNOS. DsbA-L protein expression level was determined by western blot. Results 189 T2DM (48.7±9.4 y) and 123 healthy people (42.8±8.0 y) were enrolled. In comparison with healthy control group, total adiponectin (p Conclusion This study directly demonstrated for the first time that human HMW adiponectin bioactivity was decreased in type 2 diabetes. We also provide a target for regulating HMW adiponectin concentration in diabetic patients.

Objective Adiponectin is a protein secreted predominantly by differentiated adipocytes, which plays a protective role in the development of insulin resistance and exerts beneficial actions on the hearts subjected to ischemia/reperfusion (I/R) insult. Insulin has been proved to reduce myocardial I/R injury in both normal and diabetic ones. These suggest the relationship between the insulin and adiponectin effects in diabetes. The present study aimed to investigate the effect of insulin treatment on the systemic and local cardiac adiponectin levels and on the myocardial I/R injury in type 1 diabetic mice. Methods type 1 diabetic mice were rendered through 5 days of daily intraperitoneal injection with 40 mg/kg STZ, and confirmed by markedly elevated fasting-blood glucose levels (>11.1 μmol/l). Insulin (5 IU/kg·d) was administrated for a 14, 7 or 1 day duration, respectively, since diabetic models were established. MI/R was performed by Langendorff perfusion ex vivo in hearts from the 14 day duration mice, in which the hearts were subjected to 40 min of ischemia and 60 min of reperfusion. Results The plasma adiponectin level in type 1 diabetic mice increased on the day 7 but decreased on the day 14 (p Conclusions Insulin treatment increases both plasma and cardiac adiponectin levels, which is at least partly responsible for the cardioprotective effects of insulin treatment in diabetes.

The Western meat-rich diet is both high in protein and fat. Although the hazardous effect of a high fat diet (HFD) upon liver structure and function is well recognized, whether the co-presence of high protein intake contributes to, or protects against, HF-induced hepatic injury remains unclear. Increased intake of branched chain amino acids (BCAA, essential amino acids compromising 20% of total protein intake) reduces body weight. However, elevated circulating BCAA is associated with non-alcoholic fatty liver disease and injury. The mechanisms responsible for this quandary remain unknown; the role of BCAA in HF-induced liver injury is unclear. Utilizing HFD or HFD + BCAA models, we demonstrated BCAA supplementation attenuated HFD-induced weight gain, decreased fat mass, activated mammalian target of rapamycin (mTOR), inhibited hepatic lipogenic enzymes, and reduced hepatic triglyceride content. However, BCAA caused significant hepatic damage in HFD mice, evidenced by exacerbated hepatic oxidative stress, increased hepatic apoptosis, and elevated circulation hepatic enzymes. Compared to solely HFD-fed animals, plasma levels of free fatty acids (FFA) in the HFD + BCAA group are significantly further increased, due largely to AMPKα2-mediated adipocyte lipolysis. Lipolysis inhibition normalized plasma FFA levels, and improved insulin sensitivity. Surprisingly, blocking lipolysis failed to abolish BCAA-induced liver injury. Mechanistically, hepatic mTOR activation by BCAA inhibited lipid-induced hepatic autophagy, increased hepatic apoptosis, blocked hepatic FFA/triglyceride conversion, and increased hepatocyte susceptibility to FFA-mediated lipotoxicity. These data demonstrated that BCAA reduces HFD-induced body weight, at the expense of abnormal lipolysis and hyperlipidemia, causing hepatic lipotoxicity. Furthermore, BCAA directly exacerbate hepatic lipotoxicity by reducing lipogenesis and inhibiting autophagy in the hepatocyte., Highlights • BCAA cause hepatic injury via complex mechanisms involving both adipocytes and hepatic cells. • In the adipocyte, BCAA activate AMPKα2 and stimulate lipolysis, increasing plasma free fatty acids (FFA), which in turn results in hepatic FFA accumulation. • In the liver, BCAA activate mTOR and inhibit FFA to TG conversion and autophagy, intensifying FFA lipotoxicity. High fat diet (HFD) induces systemic BCAA catabolic defects. Under HFD conditions, increased BCAA consumption further increases circulating BCAA abundance. BCAA-enhanced adipocyte lipolysis induces hyperlipidemia through activating AMPKα2. Elevated circulating FFA results in insulin resistance and hepatic lipotoxicity. Moreover, BCAA activate hepatic mTOR, inhibit lipogenesis and autophagy, therefore increasing hepatic susceptibility to FFA-mediated lipotoxicity. As BCAA are abundant in protein, our results call for caution regarding the ingestion of high protein diets in obesity and diabetic individuals, unless their BCAA metabolic pathways are determined normal.