Overexpressing STAMP2 attenuates adipose tissue angiogenesis and insulin resistance in diabetic ApoE−/−/LDLR−/− mouse via a PPARγ/CD36 pathway

The aim of this study was to investigate whether overexpression of STAMP2 improves insulin resistance by regulating angiogenesis in adipose tissues. The characteristics of diabetic mice were measured by serial metabolite and pathology tests. Samples were obtained from epididymal, subcutaneous and brown adipose tissues. Histological and morphological analysis demonstrated that STAMP2 gene overexpression reduced adipocyte size, angiogenesis in epididymal and brown adipose tissues. On aortic ring assay, microvessels sprouting from aortas were significantly inhibited after STAMP2 gene overexpression. The cellular effect of STAMP2 on angiogenesis was explored in human umbilical vein endothelial cells (HUVECs) model. Correlation of STAMP2 and angiogenesis was validated by Ad‐STAMP2 transfection and STAMP2 siRNA inhibition. In vitro, overexpression of STAMP2 significantly inhibited endothelial cell migration, tube formation. The effects of Ad‐STAMP2 transfection on HUVECs were abolished by treatment with PPARγ antagonist GW9662 (2.5 μM), and the roles of STAMP2 siRNA on HUVECs were also reversed by treatment with PPARγ agonist rosiglitazone (RSG) (0.1 mM). RT‐PCR indicated that STAMP2 could regulate levels of adhesion molecules, vascular endothelial growth factor A and CD36. The expression of PPARγ and CD36 was decreased when STAMP2 was inhibited by siRNA, while PPARγ and CD36 were highly expressed after overexpression of STAMP2. Our results suggested that STAMP2 gene overexpression may improve insulin resistance via attenuating angiogenesis in epididymal and brown adipose tissues through the PPARγ/CD36 signalling pathway.     

Lin28a protects against cardiac ischaemia/reperfusion injury in diabetic mice through the insulin‐PI3K‐mTOR pathway

The insulin‐PI3K‐mTOR pathway exhibits a variety of cardiovascular activities including protection against I/R injury. Lin28a enhanced glucose uptake and insulin‐sensitivity via insulin‐PI3K‐mTOR signalling pathway. However, the role of lin28a on experimental cardiac I/R injury in diabetic mice are not well understood. Diabetic mice underwent 30 min. of ischaemia followed by 3 hrs of reperfusion. Animals were randomized to be treated with lentivirus carrying lin28a siRNA (siLin28a) or lin28a cDNA (Lin28a) 72 hrs before coronary artery ligation. Myocardial infarct size (IS), cardiac function, cardiomyocyte apoptosis and mitochondria morphology in diabetic mice who underwent cardiac I/R injury were compared between groups. The target proteins of lin28a were examined by western blot analysis. Lin28a overexpression significantly reduced myocardial IS, improved LV ejection fraction (LVEF), decreased myocardial apoptotic index and alleviated mitochondria cristae destruction in diabetic mice underwent cardiac I/R injury. Lin28a knockdown exacerbated cardiac I/R injury as demonstrated by increased IS, decreased LVEF, increased apoptotic index and aggravated mitochondria cristae destruction. Interestingly, pre‐treatment with rapamycin abolished the beneficial effects of lin28a overexpression. Lin28a overexpression increased, while Lin28a knockdown decreased the expression of IGF1R, p‐Akt, p‐mTOR and p‐p70s6k after cardiac I/R injury in diabetic mice. Rapamycin pre‐treatment abolished the effects of increased p‐mTOR and p‐p70s6k expression exerted by lin28a overexpression. This study indicates that lin28a overexpression reduces IS, improves cardiac function, decreases cardiomyocyte apoptosis index and alleviates cardiomyocyte mitochondria impairment after cardiac I/R injury in diabetic mice. The mechanism responsible for the effects of lin28a is associated with the insulin‐PI3K‐mTOR dependent pathway.     

Overexpressing STAMP2 Improves Insulin Resistance in Diabetic ApoE?/?/LDLR?/? Mice via Macrophage Polarization Shift in Adipose Tissues

STAMP2 is a counterregulator of inflammation and insulin resistance. The aim of this study is to investigate whether activation of STAMP2 improves insulin resistance by regulating macrophage polarization in adipose tissues. The diabetic ApoE^−/−/LDLR^−/− mouse model was induced by high-fat diet and low-dose streptozotocin. Samples were obtained from epididymal, subcutaneous and brown adipose tissues. Infiltration of M1/M2 macrophages and inflammatory cytokines were investigated by immunohistochemistry. We then used gene overexpression to investigate the effect of STAMP2 on macrophages infiltration and polarization and inflammatory cytokines expression. Our results showed that infiltration of macrophages, the ratio of M1/M2 macrophages and the expression of pro-inflammatory cytokines were enhanced and STAMP2 was downregulated in adipose tissues of diabetic ApoE^−/−/LDLR^−/− mice compared with control mice. STAMP2 gene overexpression could significantly reduce macrophages infiltration, the ratio of M1/M2 macrophages and the expression of pro-inflammatory cytokines in epididymal and brown adipose tissues, improving insulin resistance. Our results suggested that STAMP2 gene overexpression may improve insulin resistance via regulating macrophage polarization in visceral and brown adipose tissues.     

Macrophage autophagy regulates mitochondria‐mediated apoptosis and inhibits necrotic core formation in vulnerable plaques

The vulnerable plaque is a key distinguishing feature of atherosclerotic lesions that can cause acute atherothrombotic vascular disease. This study was designed to explore the effect of autophagy on mitochondria‐mediated macrophage apoptosis and vulnerable plaques. Here, we generated the mouse model of vulnerable carotid plaque in ApoE^?/? mice. Application of ApoE^?/? mice with rapamycin (an autophagy inducer) inhibited necrotic core formation in vulnerable plaques by decreasing macrophage apoptosis. However, 3‐methyladenine (an autophagy inhibitor) promoted plaque vulnerability through deteriorating these indexes. To further explore the mechanism of autophagy on macrophage apoptosis, we used macrophage apoptosis model in vitro and found that 7‐ketocholesterol (7‐KC, one of the primary oxysterols in oxLDL) caused macrophage apoptosis with concomitant impairment of mitochondria, characterized by the impairment of mitochondrial ultrastructure, cytochrome c release, mitochondrial potential dissipation, mitochondrial fragmentation, excessive ROS generation and both caspase‐9 and caspase‐3 activation. Interestingly, such mitochondrial apoptotic responses were ameliorated by autophagy activator, but exacerbated by autophagy inhibitor. Finally, we found that MAPK‐NF‐κB signalling pathway was involved in autophagy modulation of 7‐KC–induced macrophage apoptosis. So, we provide strong evidence for the potential therapeutic benefit of macrophage autophagy in regulating mitochondria‐mediated apoptosis and inhibiting necrotic core formation in vulnerable plaques.     

Involvement of Yes‐associated protein 1 (YAP1) in doxorubicin‐induced cytotoxicity in H9c2 cardiac cells

Cardiac cell death is one of the major events implicated in doxorubicin‐induced cardiotoxicity, which leads to heart failure. We recently reported that Yes‐associated protein 1 (YAP1) regulates cell survival and apoptosis. However, it is unclear whether YAP1 regulates doxorubicin‐induced cell death in cardiomyocytes. We investigated whether YAP1 is involved in doxorubicin‐induced cell death using H9c2 cardiac cells and mouse heart. In an in vivo study, YAP1 protein expression was significantly decreased in hearts of doxorubicin‐treated mice with increased caspase‐3 activation. Doxorubicin also caused cell death by increasing caspase‐3 activation in H9c2 cells. Doxorubicin reduced YAP1 protein expression and messenger RNA expression accompanied by increased phosphorylation of YAP1 at Ser127. Doxorubicin further increased cell death with increased caspase‐3/7 activation in the absence of YAP1 when compared with doxorubicin or siYAP1 treatment alone. Overexpression of constitutively active YAP1 (YAP1–5SA) using an adenovirus gene transfer technique significantly reversed doxorubicin‐induced cell death by decreasing caspase‐3/7 activation in H9c2 cells. Akt, a potential prosurvival factor, decreased in doxorubicin‐ and YAP1 short interfering RNA (siRNA)‐treated cells. Doxorubicin further significantly decreased Akt protein expression when YAP1 was silenced. Overexpression of YAP1 canceled decreased Akt protein expression induced by doxorubicin treatment in H9c2 cells. In conclusion, these results suggest that doxorubicin‐induced cardiac cell death is mediated in part by down‐regulation of YAP1 and YAP1‐targeted gene, Akt. Modulating YAP1 and its related Hippo pathway on local cardiomyocytes may be a promising therapeutic approach for doxorubicin‐induced cardiotoxicity.     

Chronically elevated glucose-induced apoptosis is mediated by inactivation of Akt in cultured Müller cells

Hyperglycemia induces apoptotic cell death in a variety of cell types in diabetes, and the mechanism remains unclear. We report here that culture of rat retinal glial Müller cells in 25 mM glucose for 72 h significantly inactivated Akt and induced apoptosis. Likewise, hyperglycemia caused a significant dephosphorylation of Akt at serine-473 in Müller cells in the retina of streptozotocin-induced diabetic rats. Inactivation of Akt was associated with dephosphorylation of BAD, increased cytochrome c release, and activation of caspase-3 and caspase-9. Upregulation of Akt activity by overexpression of constitutively active Akt inhibited elevated glucose-induced apoptosis, whereas downregulation of Akt activity by overexpression of dominant negative Akt exacerbated elevated glucose-induced apoptosis, as assessed by caspase activity and nucleic acid staining. These data suggest that apoptosis induced by chronically elevated glucose is at least in part mediated by downregulation of Akt survival pathway in cultured Müller cells. It has been reported that antiapoptotic effect of Akt requires glucose in growth factor withdrawal-induced apoptosis. Our data suggest that although acutely elevated glucose may be beneficial to the cell survival, chronically elevated glucose can cause apoptosis via downregulation of Akt survival signaling.     

Tacrolimus ameliorates tubulointerstitial inflammation in diabetic nephropathy via inhibiting the NFATc1/TRPC6 pathway

Tubulointerstitial inflammation is crucial for the progression of diabetic nephropathy (DN), and tubular cells act as a driving force in the inflammatory cascade. Emerging data suggested that tacrolimus (TAC) ameliorates podocyte injury and macrophage infiltration in streptozotocin (STZ) mice. However, the effect of TAC on tubulointerstitial inflammation remains unknown. We found that albuminuria and tubulointerstitial damage improved in db/db mice treated with TAC. Macrophage infiltration and expression of IL‐6, TNF‐α, fibronectin, collagen 1 and cleaved caspase 3 were inhibited as well. In addition, the expression of nuclear factor of activated T cell 1 (NFATc1) and transient receptor potential channel 6 (TRPC6) was up‐regulated in the kidneys of DN patients and correlated with tubular injury and inflammation. The expression of NFATc1 and TRPC6 also increased in the kidneys of db/db mice and HK‐2 cells with high glucose (HG), while TAC inhibited these effects. HG‐induced inflammatory markers and apoptosis were reversed by TAC and NFATc1 siRNA in HK‐2 cells, which was abolished by TRPC6 plasmid. Furthermore, HG‐induced TRPC6 expression was inhibited by NFATc1 siRNA, while NFATc1 nuclear translocation was inhibited by TAC, but was restored by TRPC6 plasmid in HK‐2 cells under HG conditions. These findings suggest that TAC ameliorates tubulointerstitial inflammation in DN through NFATc1/TRPC6 feedback loop.     

Drak2 overexpression results in increased beta-cell apoptosis after free fatty acid stimulation

Drak2 is a serine threonine kinase in the death-associated protein family. In this study, we investigated its role in free fatty acid (FFA)-induced islet apoptosis. Drak2 mRNA and protein were rapidly induced in islet beta-cells after FFA stimulation. Such Drak2 upregulation was accompanied by increased beta-cell apoptosis, which was inhibited by Drak2 knockdown using siRNA. Conversely, transgenic (Tg) Drak2 overexpression led to aggravated beta-cell apoptosis triggered by FFA. Drak2 overexpression in islets compromised the increase of anti-apoptotic factors, such as Bcl-2, Bcl-xL and Flip, upon FFA assault. Further in vivo experiments demonstrated that Drak2 Tg mice presented compromised glucose tolerance in a diet-induced obesity model. Our data show that Drak2 is detrimental to islet survival in the presence of excessive lipid.     

Neuroprotective effects of astaxanthin against oxygen and glucose deprivation damage via the PI3K/Akt/GSK3β/Nrf2 signalling pathway in vitro

Astaxanthin (ATX), which is the most abundant flavonoid in propolis, has previously shown neuroprotective properties against cerebral ischaemia‐induced apoptosis. However, the mechanisms by which ATX mediates its therapeutic effects are unclear. At present, we explored the underlying mechanisms involved in the protective effects of ATX via the phosphoinositide 3‐kinase (PI3K)/Akt/glycogen synthase kinase 3 beta (GSK3β)/nuclear factor erythroid 2‐related factor 2 (Nrf2) signalling pathway in SH‐SY5Y cells. The PI3K/Akt inhibitor LY294002 and GSK3β inhibitor LiCl were employed in this study. Pre‐treatment with ATX for 24 hours significantly decreased the oxygen and glucose deprivation (OGD)‐induced viability loss, reduced the proportion of apoptosis and regulated OGD‐mediated reactive oxygen species (ROS) production. Furthermore, ATX suppressed OGD‐caused mitochondrial membrane potential and decomposition of caspase‐3 to cleaved caspase‐3, and heightened the B‐cell lymphoma 2 (Bcl‐2)/Bax ratio. PI3K/Akt/GSK3β/Nrf2 signalling pathway activation in SH‐SY5Y cells was verified by Western blot. ATX and LiCl treatment raised the protein levels of p‐Akt, p‐GSK3β, nucleus Nrf2 and haeme oxygenase 1 (HO‐1). However, these protein expression levels decreased by treatment of LY294002. The above in vitro data indicate that ATX can confer neuroprotection against OGD‐induced apoptosis via the PI3K/Akt/GSK3β/Nrf2 signalling pathway.     

HMGB1 mediates hyperglycaemia‐induced cardiomyocyte apoptosis via ERK/Ets‐1 signalling pathway

Apoptosis is a key event involved in diabetic cardiomyopathy. The expression of high mobility group box 1 protein (HMGB1) is up‐regulated in diabetic mice. However, the molecular mechanism of high glucose (HG)‐induced cardiomyocyte apoptosis remains obscure. We aimed to determine the role of HMGB1 in HG‐induced apoptosis of cardiomyocytes. Treating neonatal primary cardiomyocytes with HG increased cell apoptosis, which was accompanied by elevated levels of HMGB1. Inhibition of HMGB1 by short‐hairpin RNA significantly decreased HG‐induced cell apoptosis by reducing caspase‐3 activation and ratio of Bcl2‐associated X protein to B‐cell lymphoma/leukemia‐2 (bax/bcl‐2). Furthermore, HG activated E26 transformation‐specific sequence‐1 (Ets‐1), and HMGB1 inhibition attenuated HG‐induced activation of Ets‐1 via extracellular signal‐regulated kinase 1/2 (ERK1/2) signalling. In addition, inhibition of Ets‐1 significantly decreased HG‐induced cardiomyocyte apoptosis. Similar results were observed in streptozotocin‐treated diabetic mice. Inhibition of HMGB1 by short‐hairpin RNA markedly decreased myocardial cell apoptosis and activation of ERK and Ets‐1 in diabetic mice. In conclusion, inhibition of HMGB1 may protect against hyperglycaemia‐induced cardiomyocyte apoptosis by down‐regulating ERK‐dependent activation of Ets‐1.     

Nicorandil alleviates apoptosis in diabetic cardiomyopathy through PI3K/Akt pathway

Nicorandil exerts myocardial protection through its antihypoxia and antioxidant effects. Here, we investigated whether it plays an anti‐apoptotic role in diabetic cardiomyopathy. Sprague‐Dawley rats were fed with high‐fat diet; then single intraperitoneal injection of streptozotocin was performed. Rats with fasting blood glucose (FBG) higher than 11.1 mmol/L were selected as models. Eight weeks after the models were built, rats were treated with nicorandil (7.5 mg/kg day and 15 mg/kg day respectively) for 4 weeks. H9c2 cardiomyocytes were treated with nicorandil and then stimulated with high glucose (33.3 mmol/L). TUNEL assay and level of bcl‐2, bax and caspase‐3 were measured. 5‐HD was used to inhibit nicorandil. Also, PI3K inhibitor (Miltefosine) and mTOR inhibitor (rapamycin) were used to inhibit PI3K/Akt pathway. The results revealed that nicorandil (both 7.5 mg/kg day and 15mg/kg day) treatment can increase the level of NO in the serum and eNOS in the heart of diabetic rats compared with the untreated diabetic group. Nicorandil can also improve relieve cardiac dysfunction and reduce the level of apoptosis. In vitro experiments, nicorandil (100 µmol) can attenuate the level of apoptosis stimulated by high glucose significantly in H9C2 cardiomyocyte compared with the untreated group. The effect of nicorandil on apoptosis was blocked by 5‐HD, and it was accompanied with inhibition of the phosphorylation of PI3K, Akt, eNOS, and mTOR. After inhibition of PI3K/Akt pathway, the protective effect of nicorandil is restrained. These results verified that as a NO donor, nicorandil can also inhibit apoptosis in diabetic cardiomyopathy which is mediated by PI3K/Akt pathway.     

Maternal diabetes up‐regulates NOX2 and enhances myocardial ischaemia/reperfusion injury in adult offspring

Offspring of diabetic mothers are at risk of cardiovascular diseases in adulthood. However, the underlying molecular mechanisms are not clear. We hypothesize that prenatal exposure to maternal diabetes up‐regulates myocardial NOX2 expression and enhances ischaemia/reperfusion (I/R) injury in the adult offspring. Maternal diabetes was induced in C57BL/6 mice by streptozotocin. Glucose‐tolerant adult offspring of diabetic mothers and normal controls were subjected to myocardial I/R injury. Vascular endothelial growth factor (VEGF) expression, ROS generation, myocardial apoptosis and infarct size were assessed. The VEGF‐Akt (protein kinase B)‐mammalian target of rapamycin (mTOR)‐NOX2 signalling pathway was also studied in cultured cardiomyocytes in response to high glucose level. In the hearts of adult offspring from diabetic mothers, increases were observed in VEGF expression, NOX2 protein levels and both Akt and mTOR phosphorylation levels as compared to the offspring of control mothers. After I/R, ROS generation, myocardial apoptosis and infarct size were all significantly higher in the offspring of diabetic mothers relative to offspring of control mothers, and these differences were diminished by in vivo treatment with the NADPH oxidase inhibitor apocynin. In cultured cardiomyocytes, high glucose increased mTOR phosphorylation, which was inhibited by the PI3 kinase inhibitor LY294002. Notably, high glucose‐induced NOX2 protein expression and ROS production were inhibited by rapamycin. In conclusion, maternal diabetes promotes VEGF‐Akt‐mTOR‐NOX2 signalling and enhances myocardial I/R injury in the adult offspring. Increased ROS production from NOX2 is a possible molecular mechanism responsible for developmental origins of cardiovascular disease in offspring of diabetic mothers.     

MicroRNA‐351 eases insulin resistance and liver gluconeogenesis via the PI3K/AKT pathway by inhibiting FLOT2 in mice of gestational diabetes mellitus

Gestational diabetes mellitus (GDM) is known as different degree glucose intolerance that is initially identified during pregnancy. MicroRNAs (miRs) may be a potential candidate for treatment of GDM. Herein, we suggested that miR‐351 could be an inhibitor in the progression of GDM via the phosphoinositide 3‐kinase/protein kinase B (PI3K/AKT) pathway. Microarray analysis was used to identify differentially expressed genes and predict miRs regulating flotillin 2 (FLOT2). Target relationship between miR‐351 and FLOT2 was verified. Gestational diabetes mellitus mice were treated with a series of mimic, inhibitor and small interfering RNA to explore the effect of miR‐351 on insulin resistance (IR), cell apoptosis in pancreatic tissues and liver gluconeogenesis through evaluating GDM‐related biochemical indexes, as well as expression of miR‐351, FLOT2, PI3K/AKT pathway‐, IR‐ and liver gluconeogenesis‐related genes. MiR‐351 and FLOT2 were reported to be involved in GDM. FLOT2 was the target gene of miR‐351. Gestational diabetes mellitus mice exhibited IR and liver gluconeogenesis, up‐regulated FLOT2, activated PI3K/AKT pathway and down‐regulated miR‐351 in liver tissues. Additionally, miR‐351 overexpression and FLOT2 silencing decreased the levels of FLOT2, phosphoenolpyruvate carboxykinase, glucose‐6‐phosphatase, fasting blood glucose, fasting insulin, total cholesterol, triglyceride, glyeosylated haemoglobin and homeostasis model of assessment for IR index (HOMA‐IR), extent of PI3K and AKT phosphorylation, yet increased the levels of HOMA for islet β‐cell function, HOMA for insulin sensitivity index and glucose transporter 2 expression, indicating reduced cell apoptosis in pancreatic tissues and alleviated IR and liver gluconeogenesis. Our results reveal that up‐regulation of miR‐351 protects against IR and liver gluconeogenesis by repressing the PI3K/AKT pathway through regulating FLOT2 in GDM mice, which identifies miR‐351 as a potential therapeutic target for the clinical management of GDM.     

SiRNA‐Mediated Down‐Regulation of CLIC4 Gene Inhibits Cell Proliferation and Accelerates Cell Apoptosis of Mouse Liver Cancer Hca‐F and Hca‐P Cells

This study explored the effects involved in silencing CLIC4 on apoptosis and proliferation of mouse liver cancer Hca‐F and Hca‐P cells. A CLIC4‐target small interfering RNA (siRNA) was designed to compound into two individual complementary oligonucleotide chains. A process of annealing and connection to a pSilencer vector was followed by transfection with Hca‐F and Hca‐P cells. Quantitative real‐time polymerase chain reaction and Western blotting techniques were used to determine CLIC4 mRNA and protein expressions. CCK8 assay and flow cytometry were employed for analysis of the survival and apoptosis rate as well as the cell cycle in an octreotide‐induced apoptosis model. Expressions of caspase 3, caspase 9, and cleaved PARP were measured using Western blotting. The CLIC4 mRNA and protein expressions in Hca‐F and Hca‐P cells transfected by pSilencer‐CLIC4 siRNA plasmid in the blank group displayed remarkably decreased levels of expression, when compared with both the control and negative control (NC) groups. Decreased survival rates and cleaved PARP expression, increased cell apoptosis rate,expressions of caspase 3 and caspase 9 in Hca‐F and Hca‐P cells were detected in groups that had been cultured in a medium containing octreotide. The pSilencer‐CLIC4 siRNA‐2 group when compared with the control and NC groups exhibited decreased survival rates, cleaved PARP expression, increased cell apoptosis rates, and increased expressions of caspase 3 and caspase 9 of Hca‐F and Hca‐P cells. The results demonstrated that siRNA‐induced down‐regulation of CLIC4 could proliferation, while in turn promoting apoptosis of mouse liver cancer Hca‐F and Hca‐P cells. J. Cell. Biochem. 119: 659–668, 2018. © 2017 Wiley Periodicals, Inc.     

TXNIP,a novel key factor to cause Schwann cell dysfunction in diabetic peripheral neuropathy,under the regulation of PI3K/Akt pathway inhibition-induced DNMT1 and DNMT3a overexpression

Diabetic peripheral neuropathy (DPN) is the most common complication of diabetes mellitus (DM) and the dysfunction of Schwann cells plays an important role in the pathogenesis of DPN. Thioredoxin-interacting protein (TXNIP) is known as an inhibitor of thioredoxin and associated with oxidative stress and inflammation. However, whether TXNIP is involved in dysfunction of Schwann cells of DPN and the exact mechanism is still not known. In this study, we first reported that TXNIP expression was significantly increased in the sciatic nerves of diabetic mice, accompanied by abnormal electrophysiological indexes and myelin sheath structure. Similarly, in vitro cultured Schwann cells TXNIP was evidently enhanced by high glucose stimulation. Again, the function experiment found that knockdown of TXNIP in high glucose-treated RSC96 cells led to a 4.12 times increase of LC3-II/LC3-I ratio and a 25.94% decrease of cleaved caspase 3/total caspase 3 ratio. Then, DNA methyltransferase (DNMT) inhibitor 5-Aza has been reported to benefit Schwann cell in DPN, and here 5-Aza treatment reduced TXNIP protein expression, improved autophagy and inhibited apoptosis in high glucose-treated RSC96 cells and the sciatic nerves of diabetic mice. Furthermore, DNMT1 and DNMT3a upregulation were found to be involved in TXNIP overexpression in high glucose-stimulated RSC96 cells. Silencing of DNMT1 and DNMT3a effectively reversed high glucose-enhanced TXNIP. Moreover, high glucose-inhibited PI3K/Akt pathway led to DNMT1, DNMT3a, and TXNIP upregulation in RSC96 cells. Knockdown of DNMT1 and DNMT3a prevented PI3K/Akt pathway inhibition-caused TXNIP upregulation in RSC96 cells. Finally, in vivo knockout of TXNIP improved nerve conduction function, increased autophagosome and LC3 expression, and decreased cleaved Caspase 3 and Bax expression in diabetic mice. Taken together, PI3K/Akt pathway inhibition mediated high glucose-induced DNMT1 and DNMT3a overexpression, leading to cell autophagy inhibition and apoptosis via TXNIP protein upregulation in Schwann cells of DPN.Subject terms: Insulin signalling, Diabetes complications, Peripheral neuropathies     

Notch signaling protects retina from nuclear factor-κB- and poly-ADP-ribose-polymerase-mediated apoptosis under high-glucose stimulation

Proliferative diabetic retinopathy, the primary cause of vision loss in adults, is one of serious microvascular complications caused by diabetes. Both poly-ADP-ribose-polymerase (PARP) and nuclear factor (NF)-κB signaling are involved in the injury process. Injury activates PARP, which in turn potentiates NF-κB activation and causes cell apoptosis. Like the NF-κB pathway, Notch1 signaling plays a key role in the regulation of cell proliferation, differentiation, and apoptosis. However, the connections between these signaling pathways are not well understood. In this study, we used both streptozotocin (STZ)-induced diabetic mice and human retinal vascular endothelial cells (HRVECs) cultured in high glucose to detect these relationships. We found that apoptosis was increased in both STZ-induced diabetic mice and high-glucose-treated HRVECs, which was due to increased activation of PARP, cleaved caspase3, and reduced expression of Notch1 and p-Akt. The results of Notch1 overexpression and knockdown indicated that Notch1 signaling participated in the interaction of PARP and p50, and inhibited PARP- and p50-mediated apoptosis directly. These phenomena could be blocked by pretreatment with the PI3K inhibitor wortmannin via reducing p-Akt levels. Thus, our study demonstrated that Notch1 signaling protects cells from PARP- and NF-κB-induced apoptosis under high glucose through the activation of Akt.