Normal Glucose Medium Research Articles

Tribbles homolog 3 is induced by high glucose and associated with apoptosis in human endothelial cells.

Tribbles homolog 3 (TRIB3) is an intracellular kinase-like molecule that modifies cellular survival and metabolism. The present study aimed to investigate the function of TRIB3 regulation in the process of high glucose-induced apoptosis in endothelial cells, with the aim of identifying a novel intervention target for the prevention and treatment of diabetes mellitus. Human umbilical vein endothelial cells (HUVECs) grown in medium with various concentrations of glucose (5.5, 10, 20, 30 and 40 mmol/l) were assessed for mRNA expression of TRIB1, TRIB2 and TRIB3 using reverse transcription quantitative polymerase chain reaction. In addition, protein expression of TRIB3 was examined using western blot analysis. Immunofluorescence staining was performed in order to determine the distribution and localization of TRIB3 in HUVECs. Furthermore, cells grown in normal (5.5 mmol/l) or high glucose (HG; 30 mmol/l) medium were subjected to TRIB3 inhibition through small interfering (si)RNA knockdown. These cells were then examined in order to determine whether TRIB3 upregulation was associated with endothelial cell apoptosis. HUVECs treated with 30 and 40 mmol/l glucose for 48 h and 72 h showed significantly lower survival rates compared with those treated with normal glucose levels. In addition, slight but not significant increases in TRIB1 and TRIB2 mRNA expression were observed in HUVECs incubated with various concentrations of glucose for different durations. By contrast, TRIB3 mRNA expression was increased 7.2-fold following incubation with HG. Western blot analysis revealed a 5.44-fold increase in TRIB3 protein levels in cells grown in HG medium for 24 h compared with those grown in normal medium. Immunostaining assays revealed a markedly higher and well-defined nucleolar fluorescence intensity for TRIB3 expression at 24 h in HG medium compared with that of the control group. Furthermore, the apoptotic rate of HG-treated TRIB3 siRNA-transfected HUVECs was significantly increased compared with that of those transfected with control siRNA In conclusion, the results of the present study suggested that TRIB3 was associated with high glucose-induced HUVECs apoptosis, which was attenuated following transfection with TRIB3 siRNA.

THE ROLE OF G-PROTEIN COUPLED RECEPTOR 43 IN HIGH GLUCOSE-INDUCED OXIDATIVE STRESS IN HUMAN BLOOD VESSELS

Hyperglycaemia is a hallmark of diabetes and has been found to activate a reactive oxygen species (ROS)-generating enzyme, NADPH oxidase 2 (Nox2), which is involved in inflammation and diabetic cardiovascular complications. G-protein coupled receptor 43 (GPCR43) has been reported recently to be expressed in various tissues; however, its role in the human vascular system remains unknown. Acetate is agonist of GPCR43. In this study we investigated the expression and redox-signalling pathway of GPCR43 in human saphenous vein sections in a cultured model of high glucose-induced Nox2 activation and vessel dysfunction. Vessels were cultured in normal (5 mM, control) or high glucose (30 mM) medium for 24 hours in the presence or absence of acetate (5 mM) to activate GPCR43. The morphological and structural integrities of the vessels were confirmed by histological examination (H & E staining). GPCR43 was detected throughout the entire vessel wall including the endothelium, smooth muscles and adventitia. Compared to control vessels, high glucose increased (33%±10%) the expression of Nox2 in the endothelium as evaluated using immunofluorescence quantification. Increased Nox2 expression was accompanied by increased ROS production, as detected by NADPH-dependent lucigenin-chemiluminescence, and an increase in the phosphorylation of redox-sensitive signalling pathways i.e. ERK1/2 (77%±18%) and JNK (99%±23%). Acetate has no significant effect on GPCR43 expression, but prevented significantly the high glucose-induced Nox2 activation and ERK1/2 and JNK phosphorylation. In conclusion, GPCR43 is highly expressed in human vasculature and may have a therapeutic potential to reduce high glucose-induced Nox2 activation and oxidative damage of vascular function in diabetes.

Effect of calcitonin gene-related peptide on anoxia-reoxygenation induced injury to neonatal rat cardiomyocytes incubated in high glucose medium

Objective To evaluate the effect of calcitonin gene-related peptide (CGRP) on anoxiareoxygenation (A/R)-induced injury to neonatal rat cardiomyocytes incubated in high glucose medium.Methods Cardiomyocytes were obtained from 1-3-day old Sprague-Dawley rats and cultured in the culture medium containing 15％ bovine calf serum and then seeded onto 6-well plates at a density of 10 × 105/ml (3 ml/well).The cells were randomly divided into 5 groups (n =9 each):normal glucose medium control group (NG group),high glucose medium group (HG group),high glucose medium + A/R group (HG+ A/R group),high glucose medium + A/R + CGRP group (HG + A/R + CGRP group),and high glucose medium + A/R + CGRP+ CGRP8-37 group (HG + A/R + CGRP + CGRP8-37 group).The cells were incubated in normal glucose (5.5 mmol/L) medium for 72 h in NG group.In HG group,the cells were incubated in high glucose (25.0 mmol/L) medium for 72 h.In HG + A/ R group,the cells were incubated in high glucose medium for 72 h and then exposed to 3 h of anoxia followed by 2 h of reoxygenation.In group HG + A/R + CGRP,the cells were incubated in high glucose medium for 72 h,CGRP (final concentration 10-8 mol/L) was then added to the culture media and the cells were incubated for 1 h and then underwent A/R.In HG + A/R + CGRP + CGRP8-37 group,the cells were incubated in high glucose medium for 72 h,CGRP (final concentration 10 8 mol/L) was then added to the culture media and the cells were incubated for 1 h and then underwent A/R.In HG + A/R + CGRP + CGRP8-37 group,the cells were incubated in high glucose medium for 72 h,CGRP8-37 (final concentration 10-8 mol/L) and CGRP8-37 (CGRP receptor antagonist,final concentration 10-7 mol/L) was then added to the culture media and the cells were incubated for 1 h and then underwent A/R.Apoptosis in cardiomyocytes was detected using TUNEL and apoptosis index (AI) was calculated.The lactate dehydrogenase (LDH) activity in the culture medium was analyzed.Results AI and LDH activity were significantly higher in HG group than in NG group,and in HG + A/R group than in HG group.Compared with HG + A/R group,AI and LDH activity were significantly decreased in HG + A/R + CGRP group,while no significant changes were found in HG + A/R + CGRP + CGRP8-37 group.Compared with HG + A/R + CGRP group,AI and LDH activity were significantly increased in HG + A/R + CGRP + CGRP8-37 group.Conclusion CGRP attenuates A/R-induced injury to neonatal rat cardiomyocytes incubated in high glucose medium via combing with CGRP receptor. Key words: Calcitonin gene-related peptide; Diabetes mellitus; Cell hypoxia; Myocytes, cardiac ; Infant, newborn

Mechanisms by which low glucose enhances the cytotoxicity of metformin to cancer cells both in vitro and in vivo.

Different cancer cells exhibit altered sensitivity to metformin treatment. Recent studies suggest these findings may be due in part to the common cell culture practice of utilizing high glucose, and when glucose is lowered, metformin becomes increasingly cytotoxic to cancer cells. In low glucose conditions ranging from 0 to 5 mM, metformin was cytotoxic to breast cancer cell lines MCF7, MDAMB231 and SKBR3, and ovarian cancer cell lines OVCAR3, and PA-1. MDAMB231 and SKBR3 were previously shown to be resistant to metformin in normal high glucose medium. When glucose was increased to 10 mM or above, all of these cell lines become less responsive to metformin treatment. Metformin treatment significantly reduced ATP levels in cells incubated in media with low glucose (2.5 mM), high fructose (25 mM) or galactose (25 mM). Reductions in ATP levels were not observed with high glucose (25 mM). This was compensated by enhanced glycolysis through activation of AMPK when oxidative phosphorylation was inhibited by metformin. However, enhanced glycolysis was either diminished or abolished by replacing 25 mM glucose with 2.5 mM glucose, 25 mM fructose or 25 mM galactose. These findings suggest that lowering glucose potentiates metformin induced cell death by reducing metformin stimulated glycolysis. Additionally, under low glucose conditions metformin significantly decreased phosphorylation of AKT and various targets of mTOR, while phospho-AMPK was not significantly altered. Thus inhibition of mTOR signaling appears to be independent of AMPK activation. Further in vivo studies using the 4T1 breast cancer mouse model confirmed that metformin inhibition of tumor growth was enhanced when serum glucose levels were reduced via low carbohydrate ketogenic diets. The data support a model in which metformin treatment of cancer cells in low glucose medium leads to cell death by decreasing ATP production and inhibition of survival signaling pathways. The enhanced cytotoxicity of metformin against cancer cells was observed both in vitro and in vivo.

The effect of FoxO1 on the proliferation of rat mesangial cells under high glucose conditions

Hyperproliferation of glomerular mesangial cells (MCs) is a major pathological characteristic in the early stage of diabetic nephropathy (DN). We have previously confirmed that forkhead transcription factor O1 (FoxO1) was significantly downregulated in both the renal cortex of DN rats and MCs cultured under high-glucose (HG) conditions, but the effects and mechanisms of FoxO1 involved in the hyperproliferation of MCs are still unclear. This study aims to investigate whether FoxO1 regulates the hyperproliferation of MCs induced under high-glucose conditions, through modulating the cyclin-dependent kinase inhibitor (CKI), p27. Lentiviral vectors of LV-constitutively active FoxO1 (CA-FoxO1) and LV-small interfering RNA (siRNA)-FoxO1 were constructed to up- and downregulate FoxO1. Similarly, LV-NC-FoxO1 was used as negative control (NC). Rat MCs were cultured in normal glucose (5.6 mM) medium, HG (30 mM) medium, HG with LV-NC-FoxO1, HG with LV-CA-FoxO1 and HG with LV-siRNA-FoxO1 for 72 h. Cell proliferation, cell cycle progression, messenger RNA and protein expression of FoxO1, p27, cyclin D1 and CDK4 were detected by methyl thiazolyl tetrazolium assay, flow cytometry, quantitative real-time polymerase chain reaction and western blotting, respectively. MCs exposed to HG medium triggered hyperproliferation of MCs. Nevertheless, overexpression of FoxO1 caused by LV-CA-FoxO1 promoted cell cycle arrest at the G0/G1 phase and attenuated proliferation, which was associated with upregulation of p27 and downregulation of cyclin D1 and CDK4. Moreover, specific degradation of FoxO1 by LV-siRNA-FoxO1 caused a decrease of p27, increase of cyclin D1 and CDK4, overrode the limited cell cycle and stimulated proliferation of MCs. Overexpression of FoxO1 caused upregulation of p27, which promoted cell cycle arrest and inhibited hyperproliferation of MCs induced by HG. Degradation of FoxO1 caused an increase in p27 and stimulated MC proliferation. These findings unveil part of the molecular mechanism of FoxO1 regulation of MC hyperproliferation induced by HG.

165 Activation of the G-protein Coupled Receptor 43 Attenuates High Glucose-induced Vascular Oxidative Stress

Vascular oxidative stress has been found to be a prominent feature underlying the vascular abnormalities of patients with insulin-resistance and type-2 diabetes. Short chain fatty acids (SCFA) i.e. acetate (NaA),...

Effects of hyperglycemia and cyclic stretching on MMP‐2 and TGF‐β in rat mesangial cells (689.8)

Previous studies have demonstrated that matrix metalloproteinase‐2 (MMP‐2) is involved in cleaving and activating several non‐extracellular matrix substrates as well such as transforming growth factor‐beta (TGF‐β) that participate in the accumulation of mesanigal extracellular matrix leading to glomerulosclerosis during diabetes‐induced renal disease. However, the mechanism by which MMP‐2 becomes activated remains unclear. Therefore, the aim of the present study was to compare the effects of hyperglycemia and cyclic stretching (FlexCell System) on the expression of MMP‐2 and TGF‐β in cultured rat mesangial cells (MCs). MCs were seeded on 6‐well flex‐bottom plates and cultured in normal glucose (NG) medium. After 24 hrs, the wells in each plate were equally divided into NG and high glucose (HG). The next day, MCs were exposed to cyclic stretching for 18 hrs. We did not observe any differences in MMP‐2 and TGF‐β expression between static NG‐ and HG‐treated MCs. However, exposure to cyclic stretching significantly increased the expression of MMP‐2 and TGF‐β protein in both NG and HG‐treated MCs in comparison to static‐treated MCs. Moreover, MMP‐2 activity was markedly increased in response to cyclic stretching regardless of whether hyperglycemia was present. These data indicate that the upregulation of MMP‐2 and TGF‐β in MCs is primarily due to cyclic stretching rather than hyperglycemia.

High glucose improves healing of periodontal wound by inhibiting proliferation and osteogenetic differentiation of human PDL cells

Periodontal ligament (PDL) cells play an important role in wound healing of periodontal tissues. Response of PDL cells' cellular activity to high-glucose concentration levels may be the key in understanding the relationship between periodontal disease and diabetes mellitus. We studied the effect of high-glucose medium on proliferation of PDL cells in vitro. PDL cells were cultured for 1, 4, 7, 10, 14 and 17 days in normal (1100 mg/l) glucose or in high (4500 mg/l) glucose medium. The 3-(4,5-dimethylithiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay for proliferation was performed. In order to evaluate the osteogenetic differentiation of human PDL cells, the cells were induced with normal- or high-glucose medium for 1, 7, 14, 21 and 28 days. The results indicated that high glucose significantly inhibited proliferation of PDL cells. Concerning the mineralised nodule formation, the percentage of calcified area to total culture dish of PDL cells in high glucose level was lower than that in normal glucose medium. The increase in alkaline phosphatase activity and collagen expression could be observed in high-glucose-containing osteogenetic factor. In conclusion, high glucose improves healing of periodontal wound by inhibiting proliferation and differentiation of PDL cells, which could explain for delayed periodontal regeneration and healing in diabetic patients.

Inhibition of high glucose-induced apoptosis by uncoupling protein 2 in human umbilical vein endothelial cells

Studies have shown that an overproduction of mitochondrial reactive oxygen species (ROS) is an initiating cause in the pathogenesis of diabetic complications. However, uncoupling protein 2 (UCP2) can protect retinal vascular endothelial cells from damage by inhibiting the overproduction of mitochondrial ROS, although the protective mechanism involved is not completely clear. This study aimed to assess the effect and mechanism of UCP2 on the apoptosis of human umbilical vein endothelial cells (HUVECs). HUVECs were cultured in normal glucose (NG, 5.5 mmol/l) or high glucose (HG, 30 mmol/l) medium in the presence or absence of UCP2(+/+) lentiviral transfection. Lentivirus-mediated UCP2 overexpression inhibited the apoptosis of HUVECs induced by HG. Treatment with HG resulted in the upregulation of caspase-3 and cytochrome c and the downregulation of Bcl-2 in vitro. Furthermore, compared with the NG group, the rate of apoptosis was significantly increased in the HG group. On day two post-infection, NG cells showed significantly greater HUVEC cell proliferation than HG cells. Notably, UCP2 overexpression inhibited these processes. Taken together, these results suggest that UCP2 promotes cell proliferation and inhibits HG-induced apoptosis in HUVECs via the Bcl-2 up‑ and downregulation of caspase-3 and cytochrome c in vitro. This may provide experimental evidence for the application of UCP2 as a new protective factor for diabetic complications, such as diabetic retinopathy.

High Glucose Induced Oxidative Stress and Apoptosis in Cardiac Microvascular Endothelial Cells Are Regulated by FoxO3a

AimCardiac microvascular endothelial cells (CMECs) dysfunction contributes to cardiovascular complications in diabetes, whereas, the underlying mechanism is not fully clarified. FoxO transcription factors are involved in apoptosis and reactive oxygen species (ROS) production. Therefore, the present study was designed to elucidate the potential role of FoxO3a on the CMECs injury induced by high glucose.Materials and MethodsCMECs were isolated from hearts of adult rats and cultured in normal or high glucose medium for 6 h, 12 h and 24 h respectively. To down-regulate FoxO3a expression, CMECs were transfected with FoxO3a siRNA. ROS accumulation and apoptosis in CMECs were assessed by dihydroethidine (DHE) staining and TUNEL assay respectively. Moreover, the expressions of Akt, FoxO3a, Bim and BclxL in CMECs were assessed by Western blotting assay.ResultsROS accumulation in CMECs was significantly increased after high glucose incubation for 6 to 24 h. Meanwhile, high glucose also increased apoptosis in CMECs, correlated with decreased the phosphorylation expressions of Akt and FoxO3a. Moreover, high glucose incubation increased the expression of Bim, whereas increased anti-apoptotic protein BclxL. Furthermore, siRNA target FoxO3a silencing enhanced the ROS accumulation, whereas suppressed apoptosis in CMECs. FoxO3a silencing also abolished the disturbance of Bcl-2 proteins induced by high glucose in CMECs.ConclusionOur data provide evidence that high glucose induced FoxO3a activation which suppressed ROS accumulation, and in parallel, resulted in apoptosis of CMECs.

Macrophages directly mediate diabetic renal injury

Monocyte/macrophage recruitment correlates strongly with the progression of renal impairment in diabetic nephropathy (DN), yet their direct role is not clear. We hypothesized that macrophages contribute to direct podocyte injury and/or an abnormal podocyte niche leading to DN. Experiments were conducted in CD11b-DTR mice treated with diphtheria toxin (DT) to deplete macrophages after streptozotocin-induced diabetes. Additional experiments were conducted in bone marrow chimeric (CD11b-DTR→ C57BL6/J) mice. Diabetes was associated with an increase in the M1-to-M2 ratio by 6 wk after the induction of diabetes. Macrophage depletion in diabetic CD11b-DTR mice significantly attenuated albuminuria, kidney macrophage recruitment, and glomerular histological changes and preserved kidney nephrin and podocin expression compared with diabetic CD11b-DTR mice treated with mutant DT. These data were confirmed in chimeric mice indicating a direct role of bone marrow-derived macrophages in DN. In vitro, podocytes grown in high-glucose media significantly increased macrophage migration compared with podocytes grown in normal glucose media. In addition, classically activated M1 macrophages, but not M2 macrophages, induced podocyte permeability. These findings provide evidence showing that macrophages directly contribute to kidney injury in DN, perhaps by altering podocyte integrity through the proinflammatory M1 subset of macrophages. Attenuating the deleterious effects of macrophages on podocytes could provide a new therapeutic approach to the treatment of DN.

Abstract 563: Enhanced Renal Angiotensinogen Upregulation Under High Glucose Condition is Mediated by TRPA1

Clinical and basic studies have revealed that the renin-angiotensin system (RAS) plays crucial roles in the development of kidney injury during progression of type 1 diabetes mellitus (T1DM) whereas the activity of systemic RAS is suppressed in T1DM. Thus, intrarenal RAS is increasingly being recognized as an important factor in the development of diabetic nephropathy. We recently demonstrated expression of TRPA1, an oxidative stress-sensitive calcium channel, in renal proximal tubular cells (PTC) and TRPA1 mediates angiotensinogen (AGT) augmentation in the cells. However, regulation and function of the intrarenal TRPA1 in T1DM have not been elucidated. Therefore, this study was performed to evaluate intrarenal TRPA1 expression and its contribution to AGT augmentation under high glucose condition. Streptozotocin (STZ, 200 mg/kg) was intraperitoneally injected to male C57BL/6 mice. Intrarenal TRPA1 expression, kidney AGT, urinary AGT, urinary 8-isoprostane, a maker of oxidative stress, and blood glucose levels were determined one week after STZ injection. Furthermore, contribution of TRPA1 to AGT regulation was tested by using mouse PTC which was cultured with normal (1.0 g/L) or high (4.5 g/L) glucose media. Under these conditions, PTC was treated with 100 μM hydrogen peroxide. Blood glucose and urinary 8-isoprostane levels were higher in the STZ-treated mice compared with control mice. TRPA1 mRNA levels were augmented in the kidney of STZ-treated mice (2.26 ± 0.2, ratio to control mice) accompanied by increases in kidney AGT and urinary AGT levels. TPRA1 upregulation was also observed in PTC which was cultured with the high glucose medium (1.55 ± 0.11, ratio to the normal glucose). Hydrogen peroxide stimulated AGT expression in PTC. The AGT elevation was enhanced in the cultured cells with the high glucose (1.52 ± 0.13-fold in the normal glucose medium vs. 1.97 ± 0.29-fold in the high glucose medium). Treatment with a TRPA1 antagonist attenuated the enhanced AGT augmentation in PTC. These results demonstrate that intrarenal TRPA1 mediates enhanced AGT upregulation under high glucose condition and suggest that intrarenal TRPA1 is implicated in intrarenal RAS activation leading to the development of T1DM-associated diabetic nephropathy.

Abstract 338: Thioredoxin-Interacting Protein Contributes to Aggravated Myocardial Ischemia/Reperfusion Injury by Hyperglycemia

Hyperglycemia during acute myocardial infarction is common and associated with increased mortality. Thioredoxin-interacting protein (Txnip) is a modulator of cellular redox state and contributes to cell apoptosis. This study aimed to investigate whether hyperglycemia enhances Txnip expression and consequently exacerbates myocardial ischemia/reperfusion (MI/R) injury. Adult male SD rats were subjected to MI/R (30 min/4 h) and treated with saline or high glucose (HG, 500 g/L, 4 ml/kg/h, intravenously throughout the whole MI/R). In vitro study was performed on cultured neonatal rat cardiomyocytes subjected to simulated ischemia/reperfusion (SI/R) and incubated with HG (25 mM) or normal glucose (5.6 mM) medium. In vivo HG infusion significantly reduced the ± LV d P /dt max by 13.2 and 14.1% respectively (n=8, P <0.05), increased infarct size and myocardial apoptosis ( P <0.05) and increased superoxide accumulation ( P <0.01) compared with those in the saline group. Meanwhile, Txnip expression was enhanced (Ratio of Txnip/β-actin: 1.22 ± 0.16 vs. 0.92 ± 0.05 of MI/R+saline, n=6-8, P <0.05) whereas thioredoxin activity was inhibited (0.50 ± 0.05 vs. 1.41 ± 0.06 μmol/min/mg protein, P <0.01) following HG treatment in MI/R hearts. Additionally, HG significantly activated p38 MAPK and inhibited Akt in I/R hearts ( P <0.05). In cultured cardiomyocytes subjected to SI/R, HG incubation stimulated Txnip expression and reduced thioredoxin activity (n=6, P <0.05). Overexpression of Txnip enhanced HG-induced superoxide generation and aggravated cell apoptosis (n=6, P <0.05), while Txnip RNAi significantly blunted the deleterious effects of HG ( P <0.05). Moreover, inhibition of p38 MAPK or activation of Akt markedly blocked HG-induced Txnip expression in I/R cardiomyocytes. Most importantly, intramyocardial injection of Txnip siRNA markedly decreased Txnip expression and alleviated MI/R injury as evidenced by reduced infarction size (23.7 ± 1.6% vs. 43.1 ± 4.8%, n=6, P <0.05) and caspase 3 activity ( P <0.05) in HG-treated rats. Hyperglycemia enhances myocardial Txnip expression, possibly through reciprocally modulating p38 MAPK and Akt activation, leading to aggravated oxidative stress and subsequent amplification of cardiac injury following MI/R.

Inhibition of protein kinase C βII isoform rescues glucose toxicity-induced cardiomyocyte contractile dysfunction: Role of mitochondria

AimsHyperglycemia leads to cytotoxicity in the heart. Although theories were postulated for glucose toxicity-induced cardiomyocyte dysfunction including oxidative stress, the mechanism involved still remains unclear. Recent evidence has depicted a role of protein kinase C (PKC) in diabetic complications while high concentrations of glucose stimulate PKC. This study examined the role of PKCβII in glucose toxicity-induced cardiomyocyte contractile and intracellular Ca2+ aberrations. Main methodsAdult rat cardiomyocytes were maintained in normal (NG, 5.5mM) or high glucose (HG, 25.5mM) medium for 12h. Contractile and intracellular Ca2+ properties were measured using a video edge-detection system including peak shortening (PS), maximal velocity of shortening/relengthening (±dL/dt), time-to-PS (TPS), time-to-90% relengthening (TR90), rise in intracellular Ca2+ Fura-2 fluorescence intensity and intracellular Ca2+ decay. Production of ROS/O2− and mitochondrial integrity were examined using fluorescence imaging, aconitase activity and Western blotting. Key findingsHigh glucose triggered abnormal contractile and intracellular Ca2+ properties including reduced PS, ±dL/dt, prolonged TR90, decreased electrically-stimulated rise in intracellular Ca2+ and delayed intracellular Ca2+ clearance, the effects of which were ablated by the PKCβII inhibitor LY333531. Inhibition of PKCβII rescued glucose toxicity-induced generation of ROS and O2−, apoptosis, cell death and mitochondrial injury (reduced aconitase activity, UCP-2 and PGC-1α). In vitro studies revealed that PKCβII inhibition-induced beneficial effects were mimicked by the NADPH oxidase inhibitor apocynin and were canceled off by mitochondrial uncoupling using FCCP. SignificanceThese findings suggest the therapeutic potential of specific inhibition of PKCβII isoform in the management of hyperglycemia-induced cardiac complications.

177 THE EFFECTS OF SHORT CHAIN FATTY ACIDS ON HIGH LEVELS OF GLUCOSE-INDUCED ENDOTHELIAL OXIDATIVE STRESS

Hyperglycaemia is a hallmark of type 2 diabetes and plays a prominent role in oxidative stress-induced endothelial cell dysfunction and the development of cardiovascular diseases. Recently, a diet enriched with short-chain fatty acids (SCFAs) i.e. acetate, propionate and butyrate, has been found to improve insulin sensitivity and reduce blood pressure in clinical studies; however, the underlying mechanism remains elusive. In this study we investigated the effect of SCFAs on high glucose-induced endothelial dysfunction <i>in vitro</i>. Human pulmonary microvascular endothelial cells (HPMECs) were pre-incubated with acetate (5mM), propionate (4mM) or butyrate (4mM) for 1 hour before adding high glucose (30mM) and cultured for 24 h in the presence of SCFAs. Compared to cells cultured in the normal glucose medium (5mM), high glucose induced a significant increase (48.5±12.6%; p&lt;0.05) in endothelial reactive oxygen species (ROS) production as measured by both NADPH-dependant lucigenin-chemiluminescence and DHE fluorescence. Increased ROS production was accompanied with a significant increase in cell death (30.66±8.3%; p&lt;0.05) as measured by both MTT and trypan blue exclusion assays, and capillary structure breakdown of HPMEC cultured on the metrigels. We then examined the effects of SCFAs on endothelial ROS production, and found that SCFAs slightly increased the levels of ROS production in HPMEC cultured in 5mM glucose medium. However, in the presence of high glucose, SCFAs significantly inhibited high glucose-induced ROS production, reduced cell death and preserved capillary structure on the metrigels. In conclusion, SCFAs inhibit high glucose-mediated endothelial oxidative stress and improve endothelial cell function. SCFAs may represent an effective therapeutic strategy as a food supplement for cardiovascular complications linked to hyperglycaemia and diabetes.

DNA-PK Phosphorylation of IGFBP-3 Is Required to Prevent Apoptosis in Retinal Endothelial Cells Cultured in High Glucose

The goal of this study was to determine whether Compound 49b stimulates insulin-like growth factor binding protein-3 (IGFBP-3) activation in retinal endothelial cells (REC) through DNA-dependent protein kinase (DNA-PK). REC were grown in a normal glucose (5 mM) or high glucose medium (25 mM). Some cells were transfected with protein kinase A (PKA) siRNA, following treatment with 50 nM Compound 49b, a novel β-adrenergic receptor agonist. Cell proteins were extracted and analyzed for DNA-PK expression by Western blotting. Additional cells were treated with or without NU7441 (a specific DNA-PK inhibitor) prior to Compound 49b treatment. Cell lysates were processed for IGFBP-3 ELISA analyses and Western blotting to measure casein kinase 2 (CK2). Immunoprecipitation for total and phospho-IGFBP-3, cell proliferation and cell death measurements were done after transfection with the S(156)A IGFBP-3 mutation (key phosphorylation site involved in DNA-PK) plasmid DNA. Compound 49b required DNA-PK to activate IGFBP-3 in REC. IGFBP-3 activation was significantly reduced following treatment with either the DNA-PK inhibitor or following transfection with the IGFBP-3 S(156)A mutant plasmid (P < 0.05). Significant increases in cell death and decreases in cell proliferation were also observed in cells transfected with the IGFBP-3 S(156)A mutant plasmid (P < 0.05). Casein kinase levels were not altered after treatment with NU7741 or Compound 49b. Our findings suggest Compound 49b induces DNA-PK levels through PKA activity. DNA-PK is required for Compound 49b-induced IGFBP-3 expression, leading to inhibition of REC cell death.

Glucagon-Like Peptide-1 Protects Against Cardiac Microvascular Injury in Diabetes via a cAMP/PKA/Rho-Dependent Mechanism

Impaired cardiac microvascular function contributes to cardiovascular complications in diabetes. Glucagon-like peptide-1 (GLP-1) exhibits potential cardioprotective properties in addition to its glucose-lowering effect. This study was designed to evaluate the impact of GLP-1 on cardiac microvascular injury in diabetes and the underlying mechanism involved. Experimental diabetes was induced using streptozotocin in rats. Cohorts of diabetic rats received a 12-week treatment of vildagliptin (dipeptidyl peptidase-4 inhibitor) or exenatide (GLP-1 analog). Experimental diabetes attenuated cardiac function, glucose uptake, and microvascular barrier function, which were significantly improved by vildagliptin or exenatide treatment. Cardiac microvascular endothelial cells (CMECs) were isolated and cultured in normal or high glucose medium with or without GLP-1. GLP-1 decreased high-glucose–induced reactive oxygen species production and apoptotic index, as well as the levels of NADPH oxidase such as p47phox and gp91phox. Furthermore, cAMP/PKA (cAMP-dependent protein kinase activity) was increased and Rho-expression was decreased in high-glucose–induced CMECs after GLP-1 treatment. In conclusion, GLP-1 could protect the cardiac microvessels against oxidative stress, apoptosis, and the resultant microvascular barrier dysfunction in diabetes, which may contribute to the improvement of cardiac function and cardiac glucose metabolism in diabetes. The protective effects of GLP-1 are dependent on downstream inhibition of Rho through a cAMP/PKA-mediated pathway.

Effects of Aloe vera components on the Renin‐Angiotensin System in human mesangial cells

The local Renin‐Angiotensin System (RAS) is related to the development of diabetic nephropathy. Studies show that Aloe vera has hipoglicemic and antioxidant effects. Aloe emodin (AE), a component of A. vera, increases glycogen synthesis and also glucose transport via the PKI3 intracellular pathway. We evaluate if the Aloe vera components have effects on angiotensin I‐converting enzyme (ACE) activity in human mesangial cells exposed to normal (NG) and high glucose (HG) medium. Two components, P9 and P9a, were semi‐purified from A. vera by High Performance Liquid Chromatography (HPLC) and were eluated at the same retention time of the standard AE. Cells were treated with insulin, P9, P9a and AE at 1, 100 and 1000 pg/mL and ACE activity was evaluated using Z‐Phe‐His‐Leu‐OH as substrate. All treatments were compared to control. Cells under NG treated with P9, P9a and AE showed decreased ACE activity. In HG, high doses of P9 increased ACE activity and AE tend to present the same profile. These data suggest that the semi‐purified fractions contain the Aloe emodin component. Further studies are needed to elucidate the role of A. vera over RAS under physiological and pathological conditions, which may occur by sinergistic or isolated compound action. It could be important in A. vera terapeutic effects research, for instance, in diabetic nephropathy.

Urotensin II receptor antagonism confers vasoprotective effects in diabetes associated atherosclerosis: studies in humans and in a mouse model of diabetes

The small, highly conserved vasoactive peptide urotensin II (UII) is upregulated in atherosclerosis. However, its effects in diabetes-associated atherosclerosis have not been assessed. Endothelial cells were grown in normal- and high-glucose (5 and 25 mmol/l) media with and without UII (10⁻⁸ mol/l) and/or the UII receptor antagonist, SB-657510 (10⁻⁸ mol/l). Apoe knockout (KO) mice with or without streptozotocin-induced diabetes were treated with or without SB-657510 (30 mg kg⁻¹ day⁻¹; n = 20 per group) and followed for 20 weeks. Carotid endarterectomy specimens from diabetic and non-diabetic humans were also evaluated. In high (but not normal) glucose medium, UII significantly increased CCL2 (encodes macrophage chemoattractant protein 1 [MCP-1]) gene expression (human aortic endothelial cells) and increased monocyte adhesion (HUVECs). UII receptor antagonism in diabetic Apoe KO mice significantly attenuated diabetes-associated atherosclerosis and aortic staining for MCP-1, F4/80 (macrophage marker), cyclooxygenase-2, nitrotyrosine and UII. UII staining was significantly increased in carotid endarterectomies from diabetic compared with non-diabetic individuals, as was staining for MCP-1. This is the first report to demonstrate that UII is increased in diabetes-associated atherosclerosis in humans and rodents. Diabetes-associated plaque development was attenuated by UII receptor antagonism in the experimental setting. Thus UII may represent a novel therapeutic target in the treatment of diabetes-associated atherosclerosis.

Regulation of retinal endothelial cell apoptosis through activation of the IGFBP-3 receptor

The goal of this study was to investigate whether insulin-like growth factor binding protein-3 receptor (IGFBP-3 receptor) is required for IGFBP-3 to inhibit retinal endothelial cell (REC) apoptosis. REC were grown in normal glucose (5 mM) or high glucose medium (25 mM) for 3 days. Once cells reached confluence, they were transfected with an endothelial- specific IGFBP-3 plasmid DNA (non-IGF binding; IGFBP-3 NB) at 1 μg/ml for 24 h. Cell proteins were extracted and analyzed for IGFBP-3 receptor expression by Western blotting or use in coimmunoprecipitation or co-localization experiments for detection of IGFBP-3 and IGFBP-3 receptor binding. REC were also transfected with or without IGFBP-3 receptor siRNA before IGFBP-3NB plasmid DNA transfection. Cell lysates were processed for a cell death ELISA, a cleaved caspase 3 ELISA, and Western blotting to measure key pro- and anti-apoptotic markers: Bcl-xL, Bax, Cytochrome C and Akt. The IGFBP-3 receptor is present on REC. Overexpression of IGFBP-3 in REC significantly increased protein levels of IGFBP-3 receptor (p < 0.05). Significant increases in cell death were found in cells transfected with IGFBP-3 receptor siRNA versus not treated samples (p < 0.05). Data suggest that IGFBP-3 inhibits retinal endothelial cell death through activation of an IGFBP-3 receptor in a hyperglycemic environment. This is the first demonstration of the involvement of IGFBP-3 receptor in inhibition of REC cell death. Future studies will investigate the mechanism by which IGFBP-3 receptor may inhibit retinal endothelial cell death.