Class IIa HDACs do not influence beta-cell function under normal or high glucose conditions

Relationship of endothelin-1 and NLRP3 inflammasome activation in HT22 hippocampal cells in diabetes

An increased vulnerability has been detected after ischemia/reperfusion injury in cardiomyocytes in diabetic patients. Glucagon-like peptide-1 (GLP-1) has been proven to have a notable cardioprotective effect in cardiomyocytes. However, in diabetic patients, the cardioprotective effects of GLP-1 are compromised, which is called GLP-1 resistance. β-arrestin is one of the two main downstream effectors of GLP-1 and β-arrestin signaling pathway exerts cardioprotective effects upon activation of GLP-1R. Our hypothesis is that the increased vulnerability of cardiomyocytes in diabetic patients is partly due to disruption of the β-arrestin signaling pathway. To test this, we analyzed cardiomyocyte viability and survival in high glucose and normal glucose condition after hypoxia/reoxygenation injury in vitro, additional GLP-1 was used to determine whether β-arrestin signaling pathway was involved. We also investigated the role of mitochondrial dysfunction in GLP-1 resistance. Our results showed that cardioprotective effects of GLP-1 were reduced in high glucose cultured H9C2 cells compared to normal glucose cultured H9C2, verifying the existence of GLP-1 resistance in high glucose cultured H9C2 cells. Further study suggested that β-arrestin plays a key role in GLP-1 resistance: β-arrestin expression is notably downregulated in high glucose condition and cardioprotective effects of GLP-1 can be diminished by downregulation of β-arrestin in normal glucose condition while upregulation of β-arrestin can restore cardioprotective effects of GLP-1 in high glucose condition. Then we explore how β-arrestin affects the cardioprotective effects of GLP-1 and found that β-arrestin exerts cardioprotective effects by improving mitochondria quality control via the PI3K/Akt signaling pathway. Thus, our study found out a new mechanism of GLP-1 resistance of cardiomyocytes in high glucose conditions that impaired β-arrestin expression, caused mitochondria dysfunction and eventually cell death. Our study provided a new perspective in treating myocardial ischemia/reperfusion injury in diabetic patients.

Purpose. To determine the effect of prolonged exposure to high glucose on cellular behavior of normal human corneal epithelial cells (HCEC). Methods. HCEC were cultured in medium under normal or high glucose conditions for 14 days. Proliferation was evaluated by direct cell counting and [ 3 H]thymidine incorporation. Cell cycle analysis was performed using flow cytometry. The ability of HCEC to attach to type I collagen was evaluated using a short-term colorimetric adhesion assay. The effect of high glucose on the expression of integrin a 3 ß 1 was also evaluated using flow cytometry. Results. Cell number and [ 3 H]thymidine incorporation under high glucose conditions decreased compared with those under normal glucose conditions. The cells exposed to high glucose were G 0 /G 1 than untreated cells. The adhesion ability of HCEC under high glucose conditions decreased compared to normal glucose conditions. Expression of integrin a 3 ß 1 was down-regulated under high glucose conditions. Conclusions. High glucose had deleterious effects on cellular behavior of HCEC, which might cause delayed corneal epithelial wound healing in diabetic keratopathy.

Pro-Tumorigenic Roles of Cyclin Dependent Kinase 2 and its Associated Cyclins in Cholangiocarcinoma Progression under High Glucose Condition.

BackgroundEpidemiological studies have shown direct associations between type 2 diabetes and obesity, both conditions associated with hyperglycaemia and hyperinsulinemia, and the risk of pancreatic cancer. Up to 80% of pancreatic cancer patients present with either new-onset type 2 diabetes or impaired glucose tolerance at the time of diagnosis. Recent population studies indicate that the incidence of pancreatic cancer is reduced among diabetics taking metformin. In this study, the effects of exposure of pancreatic cancer cells to high glucose levels on their growth and response to metformin were investigated.MethodsThe human pancreatic cancer cell lines AsPC-1, BxPC-3, PANC-1 and MIAPaCa-2 were grown in normal (5 mM) or high (25 mM) glucose conditions, with or without metformin. The influence by metformin on proliferation, apoptosis and the AMPK and IGF-IR signalling pathways were evaluated in vitro.ResultsMetformin significantly reduced the proliferation of pancreatic cancer cells under normal glucose conditions. Hyperglycaemia however, protected against the metformin-induced growth inhibition. The anti-proliferative actions of metformin were associated with an activation of AMP-activated protein kinase AMPKThr172 together with an inhibition of the insulin/insulin-like growth factor-I (IGF-I) receptor activation and downstream signalling mediators IRS-1 and phosphorylated Akt. Furthermore, exposure to metformin during normal glucose conditions led to increased apoptosis as measured by poly(ADP-ribose) polymerase (PARP) cleavage. In contrast, exposure to high glucose levels promoted a more robust IGF-I response and Akt activation which correlated to stimulated AMPKSer485 phosphorylation and impaired AMPKThr172 phosphorylation, resulting in reduced anti-proliferative and apoptotic effects by metformin.ConclusionOur results indicate that metformin has direct anti-tumour activities in pancreatic cancer cells involving AMPKThr172 activation and suppression of the insulin/IGF signalling pathways. However, hyperglycaemic conditions enhance the insulin/IGF-I responses resulting in an altered AMPK activation profile and prevent metformin from fully switching off the growth promoting signals in pancreatic cancer cells.

Hyperglycemia is associated with an increased risk and prevalence of cardiovascular disease (CVD), due in large part to the adverse effects of glucose on endothelial cells. Indeed, hyperglycemic conditions can damage endothelial cells, in turn, impairing endothelial vasomotor and fibrinolytic function, exacerbating inflammatory and oxidative processes and propagating a proapoptotic phenotype. In addition to these primary effects, glucose also stimulates the release of extracellular microvesicles from endothelial cells. Extracellular endothelial microvesicles (EMVs) have emerged as causative agents in vascular pathology owing to their numerical increase in disease states, including diabetes, and cellular effects. The effects of glucose‐derived EMVs on endothelial cell function is not clear. The experimental aim of this study was to determine, in vitro, the effect of glucose‐derived EMVs on endothelial cell inflammation, oxidative stress, and nitric oxide (NO) production. Human umbilical vein endothelial cells (HUVECs) were cultured (3 rd passage) and incubated with RPMI 1640 media containing 25mM D‐glucose (concentration representing a diabetic glycemic state) or 5mM D‐glucose (control, normoglycemic condition) for 48 h to generate EMVs. EMVs (CD144‐PE) were counted and isolated by flow cytometry. Thereafter, HUVECs (2 x10 6 cells/condition) were treated with EMVs (2:1 EMV:cell ratio) generated from either the high (hgEMV) or normal (ngEMV) glucose condition for 24 h. EMV release was markedly higher (~280%; P<0.05) in cells treated with high vs normal glucose (134.7±15.6 vs. 35.4±3.2 EMV/µL). hgEMVs induced significantly higher release of cytokines IL‐6 (26.6±1.5 vs. 20.7±1.6 pg/mL) and IL‐8 (40.5±4.1 vs. 26.4±2.9 pg/mL); in addition, expression of active NF‐κB p65 (Ser‐536) was higher in the hgEMV treated cells (7.6±1.0 vs. 3.4±0.5 AU; P<0.05). Intracellular ROS production was higher(~26%) in the cells treated with hgEMVs (72.5±3.4 %) vs ngEMVs (57.7±1.8%). Active endothelial nitric oxide synthase (p‐eNOS Ser1177) (10.6±1.6 vs 31.7±5.2 AU) and NO production (7.7±0.2 vs. 11.6±1.1 umol/L) were both significantly lower (~30% and 20%, respectively) in hgEMV treated cells. Blocking NF‐kB activation using IKK‐2 inhibitor IV abolished the increase in IL‐6 and IL‐8 release in response to hgEMVs, verifying hgEMV induced NF‐kB‐mediated inflammation. Inhibiting endocytosis prevented the proinflammatory, pro‐oxidative and anti‐NO effects of hgEMVs; whereas, RNase‐inactivation (RNase A/T1) of hgEMVs eliminated their negative effects on endothelial proteins regulating inflammation, oxidative stress and NO production. These data demonstrate that: 1)high glucose‐derived EMVs induce a proinflammatory, pro‐oxidative, proatherogenic endothelial phenotype; and 2) EMV internalization and intracellular delivery of RNA regulatory cargo are central mechanisms underlying these deleterious cellular effects.

1. Diacylglycerol kinase (DG kinase) is a key enzyme in vascular contraction; however, alterations of the regulatory mechanisms in vascular dysfunction are poorly understood. In this study, the effect of a novel DG kinase inhibitor, stemphone, on vascular contraction was investigated. 2. The conventional DG kinase inhibitor, 6-[2-(4-[(4-fluorophenyl)phenyl-methylene]-1-piperidinyl)ethyl]-7-methyl-5H-thiazolo [3,2-alpha] pyrimidine-5-one (R59022) (0.1-30 microm), inhibited thromboxane A(2) analogue 9,11-dideoxy-11alpha,9alpha-epoxymethanoprostaglandin F(2alpha) (U46619)-induced sustained contractions in mouse aorta and porcine coronary artery in a dose-dependent manner. Treatment with stemphone did not affect contractions in these tissues. However, stemphone significantly inhibited (>0.3 microm) U46619-induced spontaneous phasic contraction in mouse portal vein. This inhibitory effect was not detected following R59022 treatment in portal vein. Therefore, stemphone demonstrated selectivity in terms of portal vein contraction. 3. Under high glucose (22.2 mm) conditions, U46619-induced contraction was enhanced in these three types of vascular tissue. Inhibitory effects of R59022 were attenuated under these conditions; however, effects of stemphone were observed. These results indicated that stemphone could inhibit portal vein contraction under high glucose conditions, for example, diabetes. These data suggested the possibility that DG kinase may be a target of hyperportal pressure. 4. Total mass of DG was enhanced under high glucose conditions. DG was derived from incorporated glucose via de novo synthesis in the absence of phospholipase C pathway mediation. This enhanced DG under high glucose conditions activated a calcium-independent protein kinase C (PKC). This PKC was associated with calcium-independent DG kinase activation. Treatment with stemphone also inhibited calcium-independent DG kinase. These signal transduction pathways were distinguishable from a DG-PKC pathway under normal glucose conditions. 5. The present investigation suggested that stemphone selectively inhibited overcontraction of portal vein induced by high glucose levels. This phenomenon was attributable to inhibition of calcium-independent DG kinase activation that occurred under high glucose conditions mediated by both DG synthesized from glucose and calcium-independent PKC activation.

The aim of the present study was to investigate the impact of high glucose concentration on the response of human periodontal ligament fibroblasts (PDLFs) to cyclic tensile strain. Human PDLFs were incubated under normal or high glucose conditions, and then were subjected to cyclic tensile stretching (8 per cent extension, 1 Hz). Gene expression was determined by quantitative real-time polymerase chain reaction. Intracellular reactive oxygen species (ROS) were determined by the 2',7'-dichlorofluorescein-diacetate assay, activation of mitogen-activated protein kinase (MAPK) was monitored by western analysis and osteoblastic differentiation was estimated with Alizarin Red-S staining. Cyclic tensile stretching of PDLF leads to an immediate activation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), as well as to the increased expression of the transcription factor c-fos, known to regulate many osteogenesis-related genes. At later time points, the alkaline phosphatase and osteopontin genes were also upregulated. Hyperglycaemic conditions inhibited these effects. High glucose conditions were unable to increase ROS levels, but they increased the medium's osmolality. Finally, increase of osmolality mimics the inhibitory effect of hyperglycaemia on MAPK activation, c-fos and osteoblast-specific gene markers' upregulation, as well as osteogenic differentiation capacity. Our findings indicate that under high glucose conditions, human PDLFs fail to adequately respond to mechanical deformation, while their strain-elicited osteoblast differentiation ability is deteriorated. The aforementioned effects are most probably mediated by the increased osmolality under hyperglycaemic conditions.

Metformin (MET) constitutes the first-line treatment against type 2 diabetes. Growing evidence linking insulin resistance and cancer risk has expanded the therapeutic potential of MET to several cancer types. However, the oncostatic mechanisms of MET are not well understood. MET has been shown to promote the expression of progesterone receptor (PGR) and other antitumor biomarkers in patients with non-diabetic endometrial cancer (EC) and in Ishikawa EC cells cultured in normal glucose (5.5 mM) media. Therefore, the present study aimed to assess the effects of MET on EC cells under conditions simulating diabetes. Ishikawa cells treated with 10 nM 17β-estradiol (E2) and/or 100 µM MET and exposed to normal and high (17.5 mM) concentrations of glucose were evaluated for proliferative and PGR expression status. Under normal glucose conditions, MET attenuated E2-induced cell proliferation and cyclin D1 gene expression, and increased total PGR and PGR-B transcript levels. MET inhibited Ishikawa cell spheroid formation only in the absence of E2 treatment. In E2-treated cells under high glucose conditions, MET showed no effects on cell proliferation and spheroid formation, and increased total PGR but not PGR-B transcript levels. Transfection with Krüppel-like factor 9 small interfering RNA increased PGR-A transcript levels, irrespective of glucose environment. Medroxyprogesterone acetate downregulated PGR-A expression more effectively with metformin under high compared with normal glucose conditions. To evaluate the potential mechanisms underlying the targeting of PGR by MET, E2-treated cells were incubated with MET and the AMPK inhibitor Compound C, or with the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), under normal glucose conditions. Compound C abrogated the effects of MET on PGR-B while AICAR increased PGR-B transcript levels, albeit less effectively compared with MET. The present results demonstrate the glucose-dependent effects of MET on PGR-B isoform expression, which may inform the response to progestin therapy in diabetic women with EC.

Glomerular mesangial cell (GMC) proliferation and matrix expansion are pathological hallmarks of a wide range of kidney diseases, including diabetic nephropathy. Although the circulating level of peptide hormone urotensin II (UII) and kidney tissue expression of UII and UII receptors (UTR) are increased in diabetic nephropathy, it remains unclear whether UII regulates GMC growth and extracellular matrix (ECM) accumulation. In this study, we tested the hypothesis that UII-induced Ca2+ signaling controls GMC proliferation and ECM production under normal and high glucose conditions. Mouse GMCs cultured under normal glucose conditions proliferated and synthesized ECM proteins in response to stimulation by mouse UII. UII-induced GMC proliferation and ECM protein synthesis were dependent on TRPC4 channel-mediated store-operated Ca2+ entry (SOCE) and sequential activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and Ca2+/cAMP response element-binding protein (CREB) transcription factor. Under high glucose conditions, GMCs synthesized UII. Moreover, proliferation and ECM production in high glucose-challenged GMCs were attenuated by selective UTR antagonist, TRPC4 channel blocker, and CaMKII and CREB-binding protein/p300 inhibitors. These findings indicate that UII-induced SOCE via TRPC4 channels stimulates CaMKII/CREB-dependent GMC proliferation and ECM protein production. Our data also suggest that UII synthesis contributes to GMC proliferation and ECM accumulation under high glucose conditions.

Diabetes increases susceptibility to infections, including Leishmania braziliensis (Lb). Our group previously demonstrated that diabetic patients with cutaneous leishmaniasis (CL) take longer to heal lesions compared to non-diabetics. Since macrophages play a critical role in CL pathogenesis, we investigated how high glucose levels impact their response during Lb infection. Macrophages cultured in high glucose conditions showed increased parasite load than those in normal glucose conditions. The production of inflammatory mediators was similar between glucose conditions, but basal reactive oxygen species (ROS) production was elevated under high glucose conditions and remained unchanged after Lb infection, indicating glucose-induced oxidative stress does not control the parasite. In contrast, macrophages in normal glucose conditions, exhibited increased ROS production only after infection. Additionally, high glucose reduced TLR2 and TLR4 expression, which was also observed after Lb infection. TLR2/4 inhibition increased Lb infection in normal glucose conditions, mediated by TLR-dependent ROS production. However, this mechanism was absent under high glucose conditions, where elevated basal ROS production appeared TLR-independent. Biopsies from diabetic CL patients corroborated these findings, showing decreased TLR2 and TLR4 expression compared to non-diabetics. These findings suggest that high glucose levels induce oxidative stress and reduces TLR expression, impairing macrophage functions and rendering them less effective at controlling Lb infection.

The hexosamine pathway has been implicated in the induction of TGFbeta1 expression and in the pathophysiology of diabetic glomerulopathy. Glucose-induced TGFbeta1 expression is mediated by p38 mitogen-activated-protein-kinase (p38-MAPK) and this kinase is activated in the diabetic glomeruli. We examined whether the p38-MAPK is implicated in hexosamine-induced TGFbeta1 mRNA expression in human mesangial cells. GFAT overexpression induced an increase in p38-MAPK activation after 6 and 12 h incubation in normal glucose, and this was prevented by the GFAT inhibitor azaserine. Furthermore, high glucose enhanced p38-MAPK activation in GFAT tranfected cells ( p</=0.04). P38-MAPK inhibition using SB202190 (1 micro mol/l) reduced hexosamine-induced TGFbeta1 expression in normal and high glucose. The activation of the p38-MAPK was dependent on protein kinase-C. The products of the hexosamine biosynthetic pathway were increased by the addition of glucosamine or by the overexpression of the rate-limiting enzyme of the hexosamine pathway, glutamine: fructose-6-phosphate amidotransferase (GFAT). Glucosamine addition resulted in cell death. UDP-N-Acetylglucosamine, one of the major hexosamine end-products, was increased in normal (7 mmol/l) and high (25 mmol/l) glucose conditions in GFAT-transfected cells compared to control transfected cells by twofold and 1.7-fold respectively ( p</=0.04) and this was accompanied by a 1.6- and 2.3-fold increase ( p</=0.02) in TGFbeta1 mRNA expression. Addition of the GFAT inhibitor azaserine (10 micro mol/l) prevented the induction of TGFbeta1 in GFAT transfected cells. Overexpression of GFAT increases hexosamine accumulation which mediates TGFbeta1 expression via a protein kinase-C and p38-MAPK dependent mechanism. Increased glucose concentrations magnify these effects.

The effects of tolbutamide on insulin, glucagon, and somatostatin secretion were investigated in the isolated perfused pancreas from normal and diabetic rats under low (30 mg/dl), normal (100 mg/dl), and high (300 mg/dl) glucose conditions. In the normal rat pancreas, tolbutamide-induced insulin release was increased when the glucose concentration in the perfusion medium was increased from 30-300 mg/dl. Tolbutamide had an inhibitory effect on glucagon release at the low (30 mg/dl) glucose concentrations, although a stimulatory effect was observed under normoglycemic conditions. The total amount of somatostatin secretion above baseline during tolbutamide infusion was higher under the normal glucose than under the low glucose condition. However, further augmentation of somatostatin release was not found at the high glucose concentration. In the diabetic rat pancreas, insulin release was diminished and tolbutamide-induced somatostatin release was enhanced with increasing glucose concentrations. Glucagon release was stimulated at the normal glucose concentration, but inhibited temporarily at the high glucose concentration. The maximum somatostatin response in the early phase was significantly decreased in the diabetic pancreas under low and normal glycemic conditions, when expressed as an incremental change (percentage) above baseline. From these results, one can conclude: (1) tolbutamide has a stimulatory effect on the pancreatic D cell in both the normal and diabetic pancreas; (2) the early response of somatostatin is decreased in the diabetic pancreas, except under conditions of high glucose concentration; and (3) the pancreatic A cell response to tolbutamide was not uniform and was quite different from the response of the D cell.

Endothelial dysfunction is a key factor for the development of organ failure in type 2 diabetes mellitus (T2DM). While hyperglycemia is a well‐established factor in the development of microvascular complications in T2DM, specific mechanisms of endothelial damage remains equivocal. In a previous study, we observed that hyperglycemic states resulted in distinct alterations in proteins involved in metabolic and angiogenic processes in endothelial cells isolated from diabetic when compared to non‐diabetic rats. In the present study, we identified and validated diverse NFκβ‐regulated cellular functions that are involved in augmenting hyperglycemia‐induced damage in the diabetic endothelium. Bioinformatics and functional studies were carried out in the primary cardiac microvascular endothelial cells (RCMVECs) derived from the spontaneously T2DM Goto‐Kakizaki (GK) rat model in comparison to the control Wistar Kyoto (WKY) rat model under normal and hyperglycemic conditions. GK and WKY RCMVECs were cultured under both normal (NG; 4.5 mM) and high glucose (HG; 25 mM) conditions for two weeks, followed by tandem mass spectrometry (MS/MS), qPCR, western blotting, microplate‐based cell proliferation, metabolic and REDOX assays. Evidence of hyperglycemia‐induced cytotoxicity in GK RCMVECs was determined by employing various microplate‐based assays, wherein a 20% reduction in cell proliferation (p&lt;0.05) and a 33% increase in PMA‐induced ROS production (p&lt;0.05) was observed. Enrichment and pathway analyses of the proteomic tandem MS/MS and qPCR datasets, revealed induction of inflammatory, metabolic and apoptotic pathways, which are tightly regulated by NFκβ, in GK RCMVECs (HG vs NG). Platelet‐activating factor receptor (LogFC = 3.5, p &lt;0.01) and beta‐catenin (LogFC= 1.3, p &lt;0.01), both key inductors of NFκβ signaling, were significantly upregulated in GK RCMVECs (HG vs NG). Western blotting analysis revealed significant upregulation of NFκβ in GK RCMVECs (HG vs NG) (FC=1.6, p&lt;0.05) and in GK NG vs WKY NG RCMVECs (FC=2.5, p&lt;0.05), suggesting a potential increase in NFκβ activity in the diabetic endothelium. Additionally, the activity of hexokinase‐2, which is reported to be a transcriptional target of NFκβ, was observed to be reduced in GK NG vs WKY NG RCMVECs (20% reduction, p&lt;0.05). Furthermore, qPCR analysis identified an upregulation of several inflammatory transcripts that are that are regulated by NFκβ such as CXCR4 (FC=7.5, p&lt;0.01) and NLRP3 (FC=5.8, p&lt;0.01) expression in GK RCMVECs (HG vs NG). By the integration of high‐throughput tandem MS/MS analysis, bioinformatics analyses and in vitro functional assays, we identified a potential induction of NFκβ signaling in GK (HG vs NG) RCMVECs, resulting in metabolic reprogramming and a heightened inflammatory and apoptotic response.Support or Funding InformationSupport for this project has been provided by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases (K01‐DK105043 to BRH) and the Department of Biomedical Engineering at the Medical College of Wisconsin and Marquette University, Milwaukee, WI, USA.

Diabetes is often associated with increased prevalence and severity of periodontal disease. We hypothesized that gingival epithelial cells modify periodontal disease progression and predicted that hyperglycemia would activate an inflammatory response in human gingival epithelial cells (HGECs). We tested our hypothesis in immortalized HGECs (epi 4 cells) isolated from periodontal tissue and transfected with the simian virus 40 T antigen. The epi 4 cells were cultured in high (25mM, HG) and normal (6mM, NG) glucose conditions. The epi 4 cells showed increased interleukin-8 (IL-8) protein secretion and mRNA expression when cultured in HG, compared with in NG. These effects were not associated with increased cell proliferation and were not observed in a hyperosmolar control group (normal glucose with 19mM mannitol). Increased IL-8 secretion in HG was inhibited by pretreatment with an antioxidant, N-acetylcysteine, or a protein kinase C inhibitor, Ro31-8220. Hyperglycemia did not affect IL-8 secretion by gingival fibroblasts or periodontal ligament cells. In epi 4 cells, hyperglycemia also induced expression of toll-like receptor 2 (TLR2) but not TLR4. These findings suggest a potential participation of epithelial cells in periodontal disease during diabetes by evoking an excessive host inflammatory response.