Haibing Chen

Sirtuin 1–Mediated Cellular Metabolic Memory of High Glucose Via the LKB1/AMPK/ROS Pathway and Therapeutic Effects of Metformin

Cellular metabolic memory occurs in diabetic microvascular and macrovascular complications, but the underlying mechanisms remain unclear. Here, we investigate the role of sirtuin 1 (SIRT1) and metformin in this phenomenon. In bovine retinal capillary endothelial cells (BRECs) and retinas of diabetic rats, the inflammatory gene, nuclear factor-κB (NF-κB), and the proapoptotic gene, Bax, induced by hyperglycemia, remained elevated after returning to normoglycemia. BRECs with small interfering RNA–mediated SIRT1 knockdown had increased sensitivity to hyperglycemia stress, whereas SIRT1 overexpression or activation by metformin inhibited the increase of mitochondrial reactive oxygen species–mediated glyceraldehyde-3-phosphate dehydrogenase by poly (ADP-ribose) polymerase (PARP) activity through the upregulation of liver kinase B1/AMP-activated protein kinase (LKB1/AMPK), ultimately suppressing NF-κB and Bax expression. Furthermore, we showed that hyperglycemia led to PARP activation, which in turn may have downregulated SIRT1. Of importance, this study also demonstrated that metformin suppressed the “memory” of hyperglycemia stress in the diabetic retinas, which may be involved in the SIRT1/LKB1/AMPK pathway. Our data suggest that SIRT1 is a potential therapeutic target for the treatment of the cellular metabolic memory, and the use of metformin specifically for such therapy may be a new avenue of investigation in the diabetes field.

Protective Effect of Perindopril on Diabetic Retinopathy Is Associated With Decreased Vascular Endothelial Growth Factor-to-Pigment Epithelium-Derived Factor Ratio : Involvement of a Mitochondria-Reactive Oxygen Species Pathway

OBJECTIVE This study aimed to verify whether the decreased vascular endothelial growth factor (VEGF)–to–pigment epithelium–derived factor (PEDF) ratio can serve as an indicator for the protective effect of angiotensin-converting enzyme inhibitors (ACEIs) on diabetic retinopathy (DR) and to investigate the role of mitochondrial reactive oxygen species (ROS) in the downregulated VEGF-to-PEDF ratio. RESEARCH DESIGN AND METHODS Diabetic rats and control animals were randomly assigned to receive perindopril or vehicle for 24 weeks, and bovine retinal capillary endothelial cells (BRECs) were incubated with normal or high glucose with or without perindopril. VEGF, PEDF, PPARγ, and uncoupling protein-2 (UCP-2) in the rat retinas or BREC extracts were examined by Western blotting and real-time RT-PCR. The levels of VEGF and PEDF in cell culture media were examined by ELISA. Mitochondrial membrane potential (Δψm) and ROS production were assayed using JC-1 or CM-H2DCFDA. RESULTS The VEGF-to-PEDF ratio was increased in the retina of diabetic rats; perindopril lowered the increased VEGF-to-PEDF ratio in diabetic rats and ameliorated the retinal damage. In BRECs, perindopril lowered the hyperglycemia-induced elevation of VEGF-to-PEDF ratio by reducing mitochondrial ROS. We found the decreased ROS production was a result of perindopril-induced upregulation of PPARγ and UCP-2 expression and the subsequent decrease of Δψm. CONCLUSIONS It is concluded that the protective effect of ACEI on DR is associated with a decreased VEGF-to-PEDF ratio, which involves the mitochondria-ROS pathway through PPARγ-mediated changes of UCP-2. This study paves a way for future application of ACEI in treatment of DR.

The Protective Effect of Perindopril on Diabetic Retinopathy is Associated with Decreased VEGF/PEDF Ratio: Involvement of a Mitochondria-ROS Pathway

OBJECTIVE This study aimed to verify whether the decreased vascular endothelial growth factor (VEGF)–to–pigment epithelium–derived factor (PEDF) ratio can serve as an indicator for the protective effect of angiotensin-converting enzyme inhibitors (ACEIs) on diabetic retinopathy (DR) and to investigate the role of mitochondrial reactive oxygen species (ROS) in the downregulated VEGF-to-PEDF ratio. RESEARCH DESIGN AND METHODS Diabetic rats and control animals were randomly assigned to receive perindopril or vehicle for 24 weeks, and bovine retinal capillary endothelial cells (BRECs) were incubated with normal or high glucose with or without perindopril. VEGF, PEDF, PPARγ, and uncoupling protein-2 (UCP-2) in the rat retinas or BREC extracts were examined by Western blotting and real-time RT-PCR. The levels of VEGF and PEDF in cell culture media were examined by ELISA. Mitochondrial membrane potential (Δψm) and ROS production were assayed using JC-1 or CM-H2DCFDA. RESULTS The VEGF-to-PEDF ratio was increased in the retina of diabetic rats; perindopril lowered the increased VEGF-to-PEDF ratio in diabetic rats and ameliorated the retinal damage. In BRECs, perindopril lowered the hyperglycemia-induced elevation of VEGF-to-PEDF ratio by reducing mitochondrial ROS. We found the decreased ROS production was a result of perindopril-induced upregulation of PPARγ and UCP-2 expression and the subsequent decrease of Δψm. CONCLUSIONS It is concluded that the protective effect of ACEI on DR is associated with a decreased VEGF-to-PEDF ratio, which involves the mitochondria-ROS pathway through PPARγ-mediated changes of UCP-2. This study paves a way for future application of ACEI in treatment of DR.

Localized-Statistical Quantification of Human Serum Proteome Associated with Type 2 Diabetes

Background Recent advances in proteomics have shed light to discover serum proteins or peptides as biomarkers for tracking the progression of diabetes as well as understanding molecular mechanisms of the disease. Results In this work, human serum of non-diabetic and diabetic cohorts was analyzed by proteomic approach. To analyze total 1377 high-confident serum-proteins, we developed a computing strategy called localized statistics of protein abundance distribution (LSPAD) to calculate a significant bias of a particular protein-abundance between these two cohorts. As a result, 68 proteins were found significantly over-represented in the diabetic serum (p<0.01). In addition, a pathway-associated analysis was developed to obtain the overall pathway bias associated with type 2 diabetes, from which the significant over-representation of complement system associated with type 2 diabetes was uncovered. Moreover, an up-stream activator of complement pathway, ficolin-3, was observed over-represented in the serum of type 2 diabetic patients, which was further validated with statistic significance (pu200a=u200a0.012) with more clinical samples. Conclusions The developed LSPAD approach is well fit for analyzing proteomic data derived from biological complex systems such as plasma proteome. With LSPAD, we disclosed the comprehensive distribution of the proteins associated with diabetes in different abundance levels and the involvement of ficolin-related complement activation in diabetes.

Improvement of Retinal Vascular Injury in Diabetic Rats by Statins Is Associated With the Inhibition of Mitochondrial Reactive Oxygen Species Pathway Mediated by Peroxisome Proliferator–Activated Receptor γ Coactivator 1α

OBJECTIVE Mitochondrial reactive oxygen species (ROS) plays a key role in diabetic retinopathy (DR) pathogenesis. However, whether simvastatin decreases diabetes-induced mitochondrial ROS production remains uncertain. The aim of this study was to clarify the beneficial effects and mechanism of action of simvastatin against diabetes-induced retinal vascular damage. RESEARCH DESIGN AND METHODS Diabetic rats and control animals were randomly assigned to receive simvastatin or vehicle for 24 weeks, and bovine retinal capillary endothelial cells (BRECs) were incubated with normal or high glucose with or without simvastatin. Vascular endothelial growth factor (VEGF) and peroxisome proliferator–activated receptor γ coactivator 1α (PGC-1α) in the rat retinas or BRECs were examined by Western blotting and real-time RT-PCR, and poly (ADP-ribose) polymerase (PARP), and p38 MAPK were examined by Western blotting. Mitochondrial membrane potential (Δψm) and ROS production were assayed using the potentiometric dye 5,5′,6,6′- Tetrachloro1,1′,3,3′-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1) or CM-H2DCFDA fluorescent probes. RESULTS Simvastatin significantly upregulated PGC-1α (P < 0.01), subsequently decreased Δψm (P < 0.05) and ROS generation (P < 0.01), inhibited PARP activation (P < 0.01), and further reduced VEGF expression (P < 0.01) and p38 MAPK activity (P < 0.01). Those changes were associated with the decrease of retinal vascular permeability, retinal capillary cells apoptosis, and formation of acellular capillaries. CONCLUSIONS Simvastatin decreases diabetes-induced mitochondrial ROS production and exerts protective effects against early retinal vascular damage in diabetic rats in association with the inhibition of mitochondrial ROS/PARP pathway mediated by PGC-1α. The understanding of the mechanisms of action of statins has important implications in the prevention and treatment of mitochondrial oxidative stress-related illness such as DR.

Serum betatrophin concentrations are significantly increased in overweight but not in obese or type 2 diabetic individuals.

In this study, circulating serum betatrophin levels were quantitated and their relationships with insulin resistance (IR) and other metabolic parameters in Chinese subjects with varying degrees of obesity and glucose tolerance were examined.

Inhibition of JAK2/STAT3-Mediated VEGF Upregulation under High Glucose Conditions by PEDF through a Mitochondrial ROS Pathway In Vitro

PURPOSEnHyperglycemia-induced mitochondrial reactive oxygen species (ROS) production plays an important role in the development of complications of diabetes such as retinopathy. However, whether pigment epithelium-derived factor (PEDF) can decrease ROS production remains uncertain. The aim of this study was to clarify whether PEDF can decrease mitochondria-derived ROS generation and subsequently downregulate vascular endothelial growth factor (VEGF) expression; the authors also investigated the involvement of Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) in the process.nnnMETHODSnBovine retinal capillary endothelial cells (BRECs) were exposed to normal glucose (NG), H(2)O(2), or high glucose (HG) in the presence or absence of PEDF. Expression of JAK2/STAT3, VEGF, uncoupling protein (UCP)-2, and proliferator-activated receptor gamma (PPARgamma) in the BRECs was examined by Western blot analysis assay; VEGF and UCP-2 mRNA were determined by real-time RT-PCR. Mitochondrial membrane potential (Deltapsim) and ROS production were assayed using JC-1 and CM-H2DCFDA, respectively.nnnRESULTSnHG exposure caused hyperpolarization of Deltapsim and increased ROS generation in BRECs; meanwhile, like H(2)O(2), it also induced the phosphorylation of JAK2/STAT3 and increased VEGF expression; these changes were inhibited by PEDF. The authors also found that PEDF-induced ROS inhibition was a result of decreased Deltapsim, which was caused by the upregulation of PPARgamma and UCP-2 expression.nnnCONCLUSIONSnFor the first time it has been demonstrated that PEDF can decrease mitochondria-derived ROS generation and subsequently downregulate VEGF expression, possibly through inhibiting HG-induced JAK2/STAT3 activation, which may offer a promising strategy for halting the development of complications of diabetes.

Oral Supplementation with Cholecalciferol 800 IU Ameliorates Albuminuria in Chinese Type 2 Diabetic Patients with Nephropathy

Background Low vitamin D levels can be associated with albuminuria, and vitamin D analogs are effective anti-proteinuric agents. The aim of this study was to investigate differences in vitamin D levels between those with micro- and those with macroalbuminuria, and to determine whether low dose cholecalciferol increases vitamin D levels and ameliorates albuminuria. Methods Two studies were performed in which 25-OH vitamin D3 (25(OH)D3) concentrations were determined by electrochemiluminescence immunoassay: 1) a cross-sectional study of patients with type 2 diabetes mellitus (T2DM) (nu200a=u200a481) and healthy controls (nu200a=u200a78); and 2) a longitudinal study of T2DM patients with albuminuria treated with conventional doses, 800 IU, of cholecalciferol for 6 months (nu200a=u200a22), and a control group (nu200a=u200a24). Results 1) Cross-sectional study: Compared to controls and T2DM patients with normoalbuminuria, serum 25(OH)D3 concentrations were significantly lower in patients with macro-albuminuria, but not in those with micro-albuminuria. Serum 25(OH)D3 levels were independently correlated with microalbuminuria. 2) Longitudinal study: Cholecalciferol significantly decreased microalbuminuria in the early stages of treatment, in conjunction with an increase in serum 25(OH)D3 levels. Conclusions Low vitamin D levels are common in type 2 diabetic patients with albuminuria, particularly in patients with macroalbuminuria, but not in those with microalbuminuria. Conventional doses of cholecalciferol may have antiproteinuric effects on Chinese type 2 diabetic patients with nephropathy.

miR-23b-3p induces the cellular metabolic memory of high glucose in diabetic retinopathy through a SIRT1-dependent signalling pathway.

Aims/hypothesisThe mechanisms underlying the cellular metabolic memory induced by high glucose remain unclear. Here, we sought to determine the effects of microRNAs (miRNAs) on metabolic memory in diabetic retinopathy.MethodsThe miRNA microarray was used to examine human retinal endothelial cells (HRECs) following exposure to normal glucose (N) or high glucose (H) for 1xa0week or transient H for 2xa0days followed by N for another 5xa0days (H→N). Levels of sirtuin 1 (SIRT1) and acetylated-nuclear factor κB (Ac-NF-κB) were examined following transfection with miR-23b-3p inhibitor or with SIRT1 small interfering (si)RNA in the H→N group, and the apoptotic HRECs were determined by flow cytometry. Retinal tissues from diabetic rats were similarly studied following intravitreal injection of miR-23b-3p inhibitor. Chromatin immunoprecipitation (ChIP) analysis was performed to detect binding of NF-κB p65 to the potential binding site of the miR-23b-27b-24-1 gene promoter in HRECs.ResultsHigh glucose increased miR-23b-3p expression, even after the return to normal glucose. Luciferase assays identified SIRT1 as a target mRNA of miR-23b-3p. Reduced miR-23b-3p expression inhibited Ac-NF-κB expression by rescuing SIRT1 expression and also relieved the effect of metabolic memory induced by high glucose in HRECs. The results were confirmed in the retina using a diabetic rat model of metabolic memory. High glucose facilitated the recruitment of NF-κB p65 and promoted transcription of the miR-23b-27b-24-1 gene, which can be suppressed by decreasing miR-23b-3p expression.Conclusions/interpretationThese studies identify a novel mechanism whereby miR-23b-3p regulates high-glucose-induced cellular metabolic memory in diabetic retinopathy through a SIRT1-dependent signalling pathway.

Urinary pigment epithelium-derived factor as a marker of diabetic nephropathy.

Background: Pigment epithelium-derived factor (PEDF), a serine protease inhibitor, regulates extracellular matrix production in the kidney. We sought the association between urinary PEDF (uPEDF) and development of nephropathy among patients with type 2 diabetes (T2DM). Methods: Two human studies were performed in which uPEDF was determined by ELISA. These studies included (1) a cross-sectional study of T2DM (n = 228) and healthy controls (n = 49) and (2) a longitudinal study of hypertensive T2DM with microalbuminuria (MA; n = 42) treated with irbesartan for 6 months. An animal study was performed in which PEDF was measured in the kidney and urine samples of control rats, rats rendered diabetic with streptozotocin that were also fed a high-fat diet, and diabetic rats treated with irbesartan for 3 months. Results: Cross-sectional study: compared to controls, uPEDF was significantly higher in patients with diabetic nephropathy. uPEDF independently correlated with MA. In the MA group, uPEDF in patients with diabetic retinopathy was significantly higher than that in patients without diabetic retinopathy. Longitudinal study: irbesartan significantly decreased uPEDF in T2DM with MA. Animal study: in diabetic rats, increased PEDF was observed in both the urine and kidney samples. uPEDF showed a significant correlation with the expression of PEDF in the kidney. Irbesartan could significantly decrease the PEDF expression in the kidneys of diabetic rats as well as uPEDF. Conclusion: uPEDF may serve as a novel marker for screening for nephropathy among patients with T2DM and monitoring the response to therapy.