How should we monitor the cardiovascular benefit of sodium–glucose cotransporter 2 inhibition?

Background

High N-terminal pro-brain-type natriuretic peptide levels have been associated with a lower risk of type 2 diabetes mellitus (T2D). However, less is known about other cardiac stress biomarkers in this context. Here we evaluated the association of mid-regional pro-atrial natriuretic peptide (MR-proANP), C-terminal pro-arginine vasopressin (copeptin), C-terminal pro-endothelin-1 (CT-proET-1) and mid-regional pro-adrenomedullin (MR-proADM) with incident T2D and changes in glucose metabolism.

Methods

We performed a prospective cohort study using data from the population-based KORA F4/FF4 study. 1773 participants (52.3% women) with MR-proANP measurements and 960 (52.7% women) with copeptin, CT-proET-1 and MR-proADM measurements were included. We examined associations of circulating plasma levels of MR-proANP, copeptin, CT-proET-1 and MR-proADM with incident T2D, the combined endpoint of incident prediabetes/T2D and with fasting and 2 h-glucose, fasting insulin, HOMA-IR, HOMA-B and HbA1c at follow-up. Logistic and linear regression models adjusted for age, sex, waist circumference, height, hypertension, total/HDL cholesterol ratio, triglycerides, smoking, physical activity and parental history of diabetes were used to compute effect estimates.

Results

During a median follow-up time of 6.4 years (25th and 75th percentiles: 6.0 and 6.6, respectively), 119 out of the 1773 participants and 72 out of the 960 participants developed T2D. MR-proANP was inversely associated with incident T2D (odds ratio [95% confidence interval]: 0.75 [0.58; 0.96] per 1-SD increase of log MR-proANP). Copeptin was positively associated with incident prediabetes/T2D (1.29 [1.02; 1.63] per 1-SD increase of log copeptin). Elevated levels of CT-proET-1 were associated with increased HOMA-B at follow-up, while elevated MR-proADM levels were associated with increased fasting insulin, HOMA-IR and HOMA-B at follow-up. These associations were independent of previously described diabetes risk factors.

Conclusions

High plasma concentrations of MR-proANP contributed to a lower risk of incident T2D, whereas high plasma concentrations of copeptin were associated with an increased risk of incident prediabetes/T2D. Furthermore, high plasma concentrations of CT-proET-1 and MR-proADM were associated with increased insulin resistance. Our study provides evidence that biomarkers implicated in cardiac stress are associated with incident T2D and changes in glucose metabolism.

     

Noscapine protects the H9c2 cardiomyocytes of rats against oxygen–glucose deprivation/reperfusion injury

Molecular Biology Reports - Noscapine is an antitumor alkaloid derived from Papaver somniferum plants. Our previous study has demonstrated that exposure of noscapine on primary murine fetal...     

Alstiphyllanines E–H,picraline and ajmaline-type alkaloids from Alstonia macrophylla inhibiting sodium glucose cotransporter

Three new picraline-type alkaloids, alstiphyllanines E–G (1–3) and a new ajmaline-type alkaloid, alstiphyllanine H (4) were isolated from the leaves of Alstonia macrophylla together with 16 related alkaloids (5–20). Structures and stereochemistry of 1–4 were fully elucidated and characterized by 2D NMR analysis. Alstiphyllanines E and F (1 and 2) showed moderate Na⁺-glucose cotransporter (SGLT1 and SGLT2) inhibitory activity. A series of a hydroxy substituted derivatives 21–28 at C-17 of the picraline-type alkaloids have been derived as having potent SGLT inhibitory activity. 10-Methoxy-N(1)-methylburnamine-17-O-veratrate (6) exhibited potent inhibitory activity, suggesting that the presence of an ester side chain at C-17 may be important to show SGLT inhibitory activity. Structure activity relationship of alstiphyllanines on inhibitory activity of SGLT was discussed.     

NADPH oxidase-derived superoxide anion-induced apoptosis is mediated via the JNK-dependent activation of NF-κB in cardiomyocytes exposed to high glucose

Hyperglycemia-induced generation of reactive oxygen species (ROS) can lead to cardiomyocyte apoptosis and cardiac dysfunction. However, the mechanism by which high glucose causes cardiomyocyte apoptosis is not clear. In this study, we investigated the signaling pathways involved in NADPH oxidase-derived ROS-induced apoptosis in cardiomyocytes under hyperglycemic conditions. H9c2 cells were treated with 5.5 or 33 mM glucose for 36 h. We found that 33 mM glucose resulted in a time-dependent increase in ROS generation as well as a time-dependent increase in protein expression of p22(phox), p47(phox), gp91(phox), phosphorylated IκB, c-Jun N-terminal kinase (JNK) and p38, as well as the nuclear translocation of NF-kB. Treatment with apocynin or diphenylene iodonium (DPI), NADPH oxidase inhibitors, resulted in reduced expression of p22(phox), p47(phox), gp91(phox), phosphorylated IκB, c-Jun N-terminal kinase (JNK) and p38. In addition, treatment with JNK and NF-kB siRNAs blocked the activity of caspase-3. Furthermore, treatment with JNK, but not p38, siRNA inhibited the glucose-induced activation of NF-κB. Similar results were obtained in neonatal cardiomyocytes exposed to high glucose concentrations. Therefore, we propose that NADPH oxidase-derived ROS-induced apoptosis is mediated via the JNK-dependent activation of NF-κB in cardiomyocytes exposed to high glucose.     

Theaflavin 3,3′-digallate reverses the downregulation of connexin 43 and autophagy induced by high glucose via AMPK activation in cardiomyocytes

Theaflavin 3,3′-digallate (TF3), is reported to protect cardiomyocytes from lipotoxicity and reperfusion injury. However, the role of TF3 in the protection of high-glucose injury is still poorly understood. This study investigated the protective effects of TF3 on gap junctions and autophagy in neonatal cardiomyocytes (NRCMs). NRCMs preincubated with high glucose were coincubated with TF3. The expression of connexins and autophagy-related proteins was determined. The functioning of gap-junctional intercellular communication (GJIC) was measured by a dye transfer assay. Adenosine monophosphate-activated protein kinase (AMPK) activity was determined by western blot. Moreover, AMPK was activated with aminoimidazole-4-carboxamide-1-β-d -ribofuranoside (AICAR) or inhibited by AMPKα small interfering RNA (siRNA) to explore the role of AMPK in the modulation of connexin 43 (Cx43) and autophagy. Meanwhile, autophagy was activated or blocked to observe the change in Cx43 expression. It was found that the protein expression of Cx43 and autophagy-related proteins was increased in a TF3 dose- and time-dependent manner under high glucose. TF3 also recovered the reduced GJIC function induced by high glucose concentrations. TF3 activated phosphorylated AMPK in a time-dependent way. AMPKα siRNA abrogated the protection of TF3, while AICAR showed similar results compared to the TF3 treatment. Meanwhile, autophagy activation caused decreased Cx43, while cotreatment with baf A1 enhanced Cx43 expression further compared with the TF3 treatment alone under high glucose. We concluded that TF3 partly reversed the inhibition of Cx43 expression and autophagy induced by high glucose in NRCMs, partly by restoring AMPK activity. Inhibition of autophagy might be protective by preserving Cx43 expression in NRCMs stimulated by high glucose.     

C-Aryl 5a-carba-β-d-glucopyranosides as novel sodium glucose cotransporter 2 (SGLT2) inhibitors for the treatment of type 2 diabetes

C-Aryl 5a-carba-β-d-glucopyranose derivatives were synthesized and evaluated for inhibition activity against hSGLT1 and hSGLT2. Modifications to the substituents on the two benzene rings resulted in enhanced hSGLT2 inhibition activity and extremely high hSGLT2 selectivity versus SGLT1. Using the created superimposed model, the reason for the high hSGLT2 selectivity was speculated to be that additional substituents occupied a new space, in a different way than known inhibitors. Among the tested compounds, the ethoxy compound 5h with high hSGLT2 selectivity exhibited more potent and longer hypoglycemic action in db/db mice than our O-carbasugar compound (1) and sergliflozin (2), which could be explained by its improved PK profiles relative to those of the two compounds. These results indicated that 5h might be a promising drug candidate for the treatment of type 2 diabetes.     

High glucose induces rat mesangial cells proliferation and MCP-1 expression via ROS-mediated activation of NF-κB pathway,which is inhibited by eleutheroside E

Glomerular hypertrophy and extracellular matrix accumulation are early features of diabetic nephropathy (DN). High glucose-induced oxidative stress is implicated in the etiology of DN. This study aims to investigate the effect of eleutheroside E (EE) on high glucose mediated rat mesangial cells (MCs) proliferation and monocyte chemoattractant protein-1 (MCP-1) expression and the underlying mechanism. MCs proliferation was assessed by MTT assay. Reactive oxygen species (ROS) level and MCP-1 expression were evaluated by ELISA kit. The protein expression of p47, NF-κB p65, p-NF-κB p65, IκBα, p-IκBα, IKKβ and p-IKKβ were determined by Western blot. The results showed that treatment with EE markedly attenuated high glucose induced MCs proliferation and in a dose-dependent manner. Intervention with EE also significantly blocked high glucose induced intracellular ROS production by decreasing NADPH oxidase activity. Meanwhile, EE administration could effectively alleviate the high glucose-stimulated activation of NF-κB, the degradation of IκBα and the expression of MCP-1. These results demonstrate that high glucose enhances MCs proliferation and MCP-1 expression by activating the ROS/NF-κB pathway and can be inhibited by EE. Our findings provide a new perspective for the clinical treatment of DN.     

Low expression of miR-186-5p regulates cell apoptosis by targeting toll-like receptor 3 in high glucose–induced cardiomyocytes

To investigate the effect and mechanism of microRNA-186-5p (miR-186-5p) on the apoptosis in high glucose (HG)–treated cardiomyocytes. Diabetic cardiomyopathy model was established in cardiomyocytes by stimulating with HG. The expressions of miR-186-5p and toll-like receptor 3 (TLR3) were detected by quantitative polymerase chain reaction or Western blot analysis, respectively. Apoptosis was detected in HG-treated cardiomyocytes by flow cytometry and Western blot analysis. The interaction between miR-186-5p and TLR3 was explored by bioinformatics analysis and luciferase activity assay. Results showed that miR-186-5p expression was downregulated in HG-treated cardiomyocytes and its overexpression reversed HG-induced apoptosis and cleaved caspase-3 protein expression. Moreover, TLR3 was indicated as a target of miR-186-5p and regulated by miR-186-5p. Knockdown of TLR3 suppressed HG-induced apoptosis and cleaved caspase-3 protein expression. Besides, restoration of TLR3 ablated the effect of miR-186-5p on cell apoptosis. Collectively, miR-186-5p attenuated HG-induced apoptosis by regulating TLR3 in cardiomyocytes, providing novel biomarker for treatment of diabetic cardiomyopathy.     

lncRNA MALAT1 mediated high glucose–induced HK-2 cell epithelial-to-mesenchymal transition and injury

Journal of Physiology and Biochemistry - Epithelial-to-mesenchymal transition (EMT) and injury of tubular cells play critical roles in the pathogenesis of diabetic nephropathy (DN). lncRNA...     

High glucose enhances TGF-β1 expression in rat bone marrow stem cells via ERK1/2-mediated inhibition of STAT3 signaling

AimsThis study was to investigate the effect of high glucose (HG) on TGF-β1 expression and the underlying mechanisms in bone marrow stem cells.Main methodsRat bone marrow multipotent adult progenitor cells (MAPCs) were cultured in normal (5.5 mM d-glucose) and HG media (25.5 mM d-glucose) for up to 14 days. l-Glucose (20 mM plus 5.5 mM d-glucose) was used as high osmolarity control. TGF-β1 expression was evaluated using quantitative RT-PCR, ELISA, and immunofluorescence staining for its mRNA and protein level in the cells and in the conditioned media. The expression and activation of ERK1/2 and STAT3 were examined in MAPCs cultured in HG media with Western blot.Key findingsMeasurable level of TGF-β1 was detected in the cells cultured in normal media. TGF-β1 expression was substantially increased in MAPCs after 36 h of culture in HG media with over 20-fold increase in the mRNA and 5-fold increase in protein level over control. Interestingly, ERK1/2 phosphorylation was significantly increased in MAPCs cultured in HG media, while in STAT3 (Tyr705), not STAT3 (Ser727), phosphorylation was dramatically decreased. Treatment of cells with the specific MEK1 inhibitor PD98059 or U0126 suppressed ERK1/2 phosphorylation and TGF-β1 expression, and completely restored the level of STAT3 (Tyr705) phosphorylation in MAPCs cultured in HG media. Treatment of the cells with the specific STAT3 phosphorylation inhibitor AG490 significantly blocked STAT3 (Tyr705) phosphorylation and increased TGF-β1 expression without change in ERK1/2 phosphorylation in MPACs.SignificanceHG increased TGF-β1 expression through inhibition of STAT3 (Tyr705) by enhanced ERK1/2 signaling in MAPCs.     

Liquid chromatography–tandem mass spectrometry determination of human plasma 1-palmitoyl-2-hydroperoxyoctadecadienoyl-phosphatidylcholine isomers via promotion of sodium adduct formation

Accumulation of phosphatidylcholine hydroperoxide (PCOOH), a primary oxidation product of phosphatidylcholine, in blood plasma has been observed in various pathological conditions, including atherosclerosis. In this study, we investigated the use of liquid chromatography–tandem mass spectrometry (LC–MS/MS) to develop a method for accurate quantification of PCOOH (1-palmitoyl-2-hydroperoxyoctadecadienoyl-sn-glycero-3-phosphocholine, 16:0/HpODE PC), focusing on isomers such as 16:0/13-HpODE PC and 16:0/9-HpODE PC. Sodiated PCOOH ([M+Na]⁺, m/z 812) provided not only a known product ion (m/z 147) but also characteristic product ions (m/z 541 for 16:0/13-HpODE PC and m/z 388 for 16:0/9-HpODE PC). Thus, three multiple reaction monitorings (MRMs) could be performed. MRM (812/147) enabled determination of 16:0/HpODE PC, and MRM (812/541) and MRM (812/388) allowed specific measurement of 16:0/13-HpODE PC and 16:0/9-HpODE PC, respectively. By using this method, we could determine plasma PCOOH concentrations in healthy subjects and patients with angiographically significant stenosis. In healthy subject and patient plasma, the concentration of 16:0/HpODE PC was close to the sum of the concentrations of 16:0/13-HpODE PC and 16:0/9-HpODE PC. This finding shows that radical and/or enzymatic oxidation, rather than singlet oxygen oxidation, is recognized to cause peroxidation of PC. The newly developed LC–MS/MS method appears to be a powerful tool for developing a better understanding of in vivo lipid peroxidation and its involvement in human diseases.     

Sufentanil protects the rat myocardium against ischemia–reperfusion injury via activation of the ERK1/2 pathway

Sufentanil, a lipophilic opioid, is the most frequently used clinical drug for ischemic heart disease. The effects of sufentanil on MAPK signaling in ischemic heart disease were explored. The effects of sufentanil on ischemia–reperfusion (IR)-induced myocardial injury in a rat model were examined. The serum levels of CK, LDH, MDA and SOD, and the activities of Na⁺–K⁺-ATPase and Ca²⁺–Mg²⁺-ATPase were measured. The levels of total and phosphorylated ERK1/2, JNK, and p38 were measured by western blotting in the heart, and the myocardial H9C2 cell line was studied. Using the Cell Counting Kit-8, the growth rate of H9C2 cells affected by sufentanil was studied. The serum levels of CK, LDH and MDA were higher in the IR group than in the SO and SUF groups. The SOD level, as well as the activities of Na⁺–K⁺-ATPase and Ca²⁺–Mg²⁺-ATPase, were lower in the SO and SUF groups than in the IR group. The phosphorylated ERK1/2 level was lower in the IR group than in the SO and SUF groups. The growth rate of H9C2 cells increased with the concentration of sufentanil and the exposure time. The phosphorylated ERK level was upregulated by 4–12 h of sufentanil exposure, indicating that the effects were time-dependent. Furthermore, an inhibition of ERK signaling by chemical inhibition suppressed the sufentanil-mediated increase in the growth rate of H9C2 cells. Sufentanil appears to be beneficial for cases of worsening ischemic heart disease. Further studies are necessary before a clinical application is considered.     

Two-dimensional 1H NMR studies of rat atrial natriuretic factor(1–23)

Using a variety of two-dimensional NMR methods, the ¹H NMR resonances of rat ANF(1–23) in dimethyl sulfoxide-d6 solution have been assigned. Two-dimensional phase sensitive correlated spectroscopy was used to identify protons that are scalar coupled and were also used to obtain coupling constants (³J_NH-αCH) in complicated regions of the spectra. Relayed coherence transfer experiments proved useful in identifying the connectivities between the NH and β-protons of the same amino acid residue. Finally, phase sensitive 2D NOE experiments allowed the identification of protons close in space between adjacent residues, thus providing the sequential assignments as well as conformational information. These preliminary results (chemical shifts, coupling constants, NOEs) were analyzed in terms of possible polypeptide secondary structures and were found to be consistent with a β-type structure or an averaging of solution conformations (random coil).     

The mechanism of glucose 6-phosphate–d-myo-inositol 1-phosphate cyclase of rat testis. The involvement of hydrogen atoms

Comparison of the initial (3)H/(14)C ratios in specifically labelled d-glucose 6-phosphates with the final ratios in myo-inositol produced by glucose 6-phosphate-d-myo-inositol 1-phosphate cyclase from rat testis showed that, during the conversion, the hydrogen atoms at C-1 and C-3 were fully retained, one hydrogen atom was lost from C-6, and that at C-5 was apparently retained to the extent of 80-90%. The loss of (3)H could not be stimulated by addition of unlabelled NADH, and when unlabelled substrate was used (3)H from [(3)H]NADH and [(3)H]water was not incorporated. Treatment of the enzyme with charcoal abolished the activity, and this was restored to 25-50% of the original activity by NAD(+). The charcoal-treated enzyme again apparently gave 85% retention of hydrogen with [5-(3)H]glucose 6-phosphate as substrate in the presence of NAD(+) alone, but the retention was decreased to 65% with excess of NADH. The results are interpreted as indicating that the cyclization proceeds by an aldol condensation in which C-5 is oxidized by NAD(+) in a tightly-bound ternary complex, and that the apparent loss of (3)H when untreated enzyme is used is due to an isotope effect. It is suggested that after treatment with charcoal some exchange of NADH with an external pool may take place.     

α-Lipoic acid increases tolerance of cardiomyoblasts to glucose/glucose oxidase-induced injury via ROS-dependent ERK1/2 activation

α-Lipoic acid (LA) has been shown to improve the diabetic cardiac symptoms. However, the underlying mechanisms have not been elucidated precisely. We have reported recently that LA potentially protected neurons from substance-induced apoptosis. We hypothesized that LA could attenuate cardiac cells death induced by oxidative stress derived from high glucose. To test this possibility, we examined the effects of LA on d-glucose/glucose oxidase (DG/GO, 30mM/5mU)-induced injury in rat cardiomyoblast H9c2 cells. We observed that LA pretreatment significantly increased cell viability in DG/GO-challenged cells. LA pretreatment also attenuated DG/GO-induced apoptosis as evidenced by decreases in both nuclear condensation and loss of mitochondrial potential. In addition, LA activated ERK1/2 and moderately increased ROS production. Blockade of ERK1/2 activation by PD98059 completely abolished LA-induced protection against DG/GO challenge. Inhibition of ROS by N-acetylcysteine abrogated LA-induced ERK1/2 activation and cytoprotection. Furthermore, we observed that the ROS production induced by LA was significantly slower and milder than that by DG/GO. Our results suggest that pretreatment with LA moderately increased ROS production to induce a preconditioning-like effect by ERK1/2 activation thereby increased tolerance of H9c2 cells to DG/GO challenge.     

High levels of glucose induce “metabolic memory” in cardiomyocyte via epigenetic histone H3 lysine 9 methylation

Diabetic patients continue to develop inflammation and cardiovascular complication even after achieving glycemic control, suggesting a "metabolic memory". Metabolic memory is a major challenge in the treatment of diabetic complication, and the mechanisms underlying metabolic memory are not clear. Recent studies suggest a link between chromatin histone methylation and metabolic memory. In this study, we tested whether histone 3 lysine-9 tri-methylation (H3K9me3), a key epigenetic chromatin marker, was involved in high glucose (HG)-induced inflammation and metabolic memory. Incubating cardiomyocyte cells in HG resulted in increased levels of inflammatory cytokine IL-6 mRNA when compared with myocytes incubated in normal culture media, whereas mannitol (osmotic control) has no effect. Chromatin immunoprecipitation (ChIP) assays showed that H3K9me3 levels were significantly decreased at the promoters of IL-6. Immunoblotting demonstrated that protein levels of the H3K9me3 methyltransferase, Suv39h1, were also reduced after HG treatment. HG-induced apoptosis, mitochondrial dysfunction and cytochrome-c release were reversible. However, the effects of HG on the expression of IL-6 and the levels of H3K9me3 were irreversible after the removal of HG from the culture. These results suggest that HG-induced sustained inflammatory phenotype and epigenetic histone modification, rather than HG-induced mitochondrial dysfunction and apoptosis, are main mechanisms responsible for metabolic memory. In conclusion, our data demonstrate that HG increases expression of inflammatory cytokine and decreases the levels of histone-3 methylation at the cytokine promoter, and suggest that modulating histone 3 methylation and inflammatory cytokine expression may be a useful strategy to prevent metabolic memory and cardiomyopathy in diabetic patients.     

LOX-1过表达诱发H9C2心肌细胞肥大

目的:构建携带过表达大鼠凝集素样氧化低密度脂蛋白受体-1(LOX-1)基因的慢病毒载体,研究LOX-1与心肌细胞肥大的关系。方法:构建大鼠LOX-1基因pHIV-LOX-1过表达质粒,与包装质粒psPAX2、pMD2G共转染293T细胞,检测其侵染效率。包装慢病毒并侵染H9C2心肌细胞,72 h后观察其侵染效率。qPCR法检测细胞LOX-1表达。检测过表达LOX-1后心肌细胞面积及其蛋白含量变化。结果:成功构建过表达LOX-1 H9C2心肌细胞。过表达LOX-1组(Lv.LOX-1+)心肌细胞面积(16691.890±1022.368μm2)较对照组(Lv.NC)(3459.865±343.175μm2)显著增加(P0.001)。Lv.LOX-1+组心肌细胞蛋白含量(132.457±8.188 pg/cell)较Lv.NC组(45.095±1.655 pg/cell)显著增加(P0.001)。结论:LOX-1过表达能诱导H9C2心肌细胞肥大。     

[15N,1H]/[13C,1H]-TROSY for simultaneous detection of backbone 15N–1H, aromatic 13C–1H and side-chain 15N–1H2 correlations in large proteins

This paper describes a [¹⁵N,¹H]/[¹³C,¹H]-TROSY experiment for the simultaneous acquisition of the heteronuclear chemical shift correlations of backbone amide ¹⁵N–¹H groups, side chain ¹⁵N–¹H₂ groups and aromatic ¹³C–¹H groups in otherwise highly deuterated proteins. The ¹⁵N–¹H and ¹³C–¹H correlations are extracted from two subspectra of the same data set, thus preventing possible spectral overlap of aromatic and amide protons in the ¹H dimension. The side-chain ¹⁵N–¹H₂ groups, which are suppressed in conventional [¹⁵N,¹H)-TROSY, are observed with high sensitivity in the ¹⁵N–¹H subspectrum. [¹⁵N,¹H]/[¹³C,¹H]-TROSY was used as the heteronuclear correlation block in a 3D [¹H,¹H]-NOESY-[¹⁵N,¹H]/[¹³C,¹H]-TROSY experiment with the membrane protein OmpA reconstituted in detergent micelles of molecular weight 80000 Da, which enabled the detection of numerous NOEs between backbone amide protons and both aromatic protons and side chain ¹⁵N–¹H₂ groups.