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Diabetic retinopathy (DR) is one of the most common complications of diabetes worldwide and is associated with visual loss and blindness. However, effective treatments for both early- and late-stage DR remain lacking. A streptozotocin-induced diabetic mouse model and high glucose (HG)–treated Müller cell model were established. M1/M2 microglia polarization was assessed by immunofluorescence staining and flow cytometry. Expression of long noncoding RNA (lncRNA) OGRU, cytokines, and other key molecules was detected by quantitative RT-PCR or Western blot. ELISA was used to monitor cytokine secretion. Müller cell–derived exosomes were isolated and characterized by nanopartical tracking analysis, Western blot, and transmission electron microscopy, and exosome uptake assay was used to monitor the intercellular transport of exosomes. Associations among lncRNA-miRNA-mRNA networks were validated by RNA pulldown and RNA immunoprecipitation and dual luciferase assays. Increased M1 polarization but decreased M2 polarization of retinal microglia was observed in DR mice. HG-treated Müller cell–derived exosomes transported OGRU into microglia and promoted microglia polarization toward the M1 phenotype. Mechanistically, OGRU served as a competing endogenous RNA for miR-320-3p, miR-221-3p, and miR-574-5p to regulate aldose reductase (AR), PFKFB3, and glucose transporter 1 (GLUT1) expression in microglia, respectively. Loss of miR-320-3p/miR-221-3p/miR-574-5p or reinforced AR/PFKFB3/GLUT1 abrogated OGRU silencing–mediated microglia polarization in vitro. In vivo studies further showed that OGRU/miR-320-3p/AR, OGRU/miR-221-3p/PFKFB3, and OGRU/miR-574-5p/GLUT1 axes regulated microglia polarization in DR mice. Collectively, Müller cell–derived exosomal OGRU regulated microglia polarization in DR by modulating OGRU/miR-320-3p/AR, OGRU/miR-221-3p/PFKFB3, and OGRU/miR-574-5p/GLUT1 axes. Article Highlights: Retinal microglia shifted from the M2 toward the M1 phenotype in mice with diabetic retinopathy (DR). High glucose–treated Müller cell–derived exosomal OGRU promoted microglia polarization toward the M1 phenotype. OGRU served as a competing endogenous RNA for miR-320-3p, miR-221-3p, and miR-574-5p to regulate aldose reductase/PFKFB3/glucose transporter 1 expression in microglia. OGRU promoted DR progression by taking essential actions in microglia polarization. [ABSTRACT FROM AUTHOR]

In diabetic patients, excess vascular inflammation and cardiovascular disease (CVD) are the leading causes of morbidity and mortality, and remain major public health issues. Trivalent chromium is thought to be necessary for normal glucose and lipid metabolism. This study examined the effect of chromium niacinate (Cr-N) or chromium picolinate (Cr- P) on vascular inflammation markers [TNF-α., IL-6, LP, HbA[sub 1], cholesterol (chol), triglycerides (TG) in D], and on pro-inflammatory cytokines in cultured U937 monocytes. Diabetes was induced in Sprague Dawley rats by STZ. Control-buffer or Cr-N or Cr-P (400 µg Cr/Kg BW) suppl by gavages daily for 7 wks. Blood was collected by heart puncture using light anesthesia from normal (N, n=6)); diabetic (D, n=6); D+CrN (n=5); and D+Cr-P (n=5) groups of rats. D caused a significant increase in TNF-α, IL-6, LP, HbA[sub 1], chol and TG in D. Compared with D, Cr-N suppl lowered the TNF-α (7.8±2.6 vs 46.3±14 pg/ml, p=0.04), IL-6 (101±13 vs 180±43 pg/ml, 0.02) LP (0-40±.03 vs 0.95±0.14). nmol malondialdehyde/ml, p=0.01), HbA[sub 1] (9.6±0.1 vs 11.0±0.3%, p=0.02), TG (540±111 vs 1137±185mg%, p=0.04) and chol (128±6 vs 174±12mg%, (p=0.04). Compared with D, D+Cr-P showed a decrease in TNF-α (6.4±3.1 vs 46.3±14, p--0.024), IL-6 (107±9 vs 180±143 pg/ml, p=0.02) LP (0.43±0.04 vs 0.95±0.14, p--0.01) and. Thus, Cr-N suppl lowers TNF-α, IL-6, LP, HbA[sub 1], chol, and TG levels in D rats. Similarly, effect of riG on IL-6, IL-8 and MCP-1 secretion was abolished by Cr-N, and to a lesser extent by Cr-P in cultured U937 monocytes. Thus, Cr-N supplementation is more effective form of Cr[sup 3+] in preventing vascular inflammation both in a cell culture model and in diabetic rats, and thereby, can reduce the risk of CVD in diabetes. [ABSTRACT FROM AUTHOR]

Mitochondria experience genomic and functional instability in diabetes, and mitochondrial dysfunction has a critical role in the development of diabetic retinopathy. Diabetes also alters expressions of many long noncoding RNAs (LncRNAs), the RNAs with >200 nucleotides and no open reading frame. LncRNAs are mainly encoded by the nuclear genome, but mtDNA also encodes three LncRNAs. Our goal was to investigate the effect of hyperglycemia on mtDNA-encoded LncRNA cytochrome B (LncCytB) in mtDNA stability in diabetic retinopathy. Retinal endothelial cells, transfected with LncCytB-overexpressing plasmids or siRNA, incubated in 5 mmol/L d-glucose (normal glucose [NG]) or 20 mmol/L d-glucose (high glucose [HG]) for 4 days, were analyzed for LncCytB expression by strand-specific PCR and its mitochondrial localization by RNA fluorescence in situ hybridization. Damage-sensitive mtDNA regions were examined by micrococcal nuclease (MNase) digestion sequencing and LncCytB occupancy at mtDNA by chromatin isolation by RNA purification. Protective nucleoids in mtDNA were analyzed by SYBR Green-MitoTracker Red staining and confirmed in isolated mitochondria by flow cytometry. Compared with NG, HG downregulated LncCytB by >50% but had no significant effect on the other mtDNA-encoded LncRNAs. mtDNA packaging was impaired, MNase sensitivity was increased, and LncCytB occupancy at mtDNA was decreased. While LncCytB overexpression ameliorated mtDNA damage and decrease in nucleoids and copy numbers, LncCytB-siRNA exacerbated damage and further reduced nucleoids. Retinal microvessels from streptozotocin-induced diabetic mice and human donors with diabetic retinopathy presented a similar decrease in LncCytB and mtDNA nucleoids. Thus, LncCytB has a major role in maintaining mitochondrial genomic stability, and its downregulation in the hyperglycemic milieu contributes to increased vulnerability of mtDNA to damage. ARTICLE HIGHLIGHTS: In the pathogenesis of diabetic retinopathy, an mtDNA-encoded long noncoding RNA, long noncoding cytochrome B (LncCytB), is downregulated. Downregulation of LncCytB results in the reduced number of protective nucleoids, and the mtDNA becomes vulnerable to the damage. The damaged mtDNA compromises the electron transport chain system, increasing superoxide levels, and the vicious cycle of free radicals continues to self-propagate. Preventing the downregulation of LncCytB in diabetes will interfere in the self-perpetuating vicious cycle of free radicals and should inhibit the development of diabetic retinopathy. [ABSTRACT FROM AUTHOR]

Exosomes have been implicated in diabetic kidney disease (DKD), but the regulation of exosomes in DKD is largely unknown. Here, we have verified the decrease of exosome secretion in DKD and unveiled the underlying mechanism. In Boston University mouse proximal tubule (BUMPT) cells, high-glucose (HG) treatment led to a significant decrease in exosome secretion, which was associated with specific downregulation of RAB27B, a key guanosine-5'-triphosphatase in exosome secretion. Overexpression of RAB27B restored exosome secretion in HG-treated cells, suggesting a role of RAB27B downregulation in the decrease of exosome secretion in DKD. To understand the mechanism of RAB27B downregulation, we conducted bioinformatics analysis that identified FOXO1 binding sites in the Rab27b gene promoter. Consistently, HG induced phosphorylation of FOXO1 in BUMPT cells, preventing FOXO1 accumulation and activation in the nucleus. Overexpression of nonphosphorylatable, constitutively active FOXO1 led to the upregulation of RAB27B and an increase in exosome secretion in HG-treated cells. In vivo, compared with normal mice, diabetic mice showed increased FOXO1 phosphorylation, decreased RAB27B expression, and reduced exosome secretion. Collectively, these results unveil the mechanism of exosome dysfunction in DKD where FOXO1 is phosphorylated and inactivated in DKD, resulting in RAB27B downregulation and the decrease of exosome secretion. [ABSTRACT FROM AUTHOR]

Diabetic vasculopathy, encompassing complications such as diabetic retinopathy, represents a significant source of morbidity, with inflammation playing a pivotal role in the progression of these complications. This study investigates the influence of N 6-methyladenosine demethylase (m6A) modification and the m6A demethylase fat mass and obesity-associated (FTO) protein on macrophage polarization and its subsequent effects on diabetic microvasculopathy. We found that diabetes induces a shift in macrophage polarization toward a proinflammatory M1 phenotype, which is associated with a reduction in m6A modification levels. Notably, FTO emerges as a critical regulator of m6A under diabetic conditions. In vitro experiments reveal that FTO not only modulates macrophage polarization but also mediates their interactions with vascular endothelial cells. In vivo experiments demonstrate that FTO deficiency exacerbates retinal inflammation and microvascular dysfunction in diabetic retinas. Mechanistically, FTO stabilizes mRNA through an m6A-YTHDF2–dependent pathway, thereby activating the PI3K/AKT signaling cascade. Collectively, these findings position FTO as a promising therapeutic target for the management of diabetic vascular complications. Article Highlights: The fat mass and obesity-associated (FTO) protein is a key regulator for decreased N 6-methyladenosine modification in diabetes-induced macrophage polarization. FTO not only modulates macrophage polarization but also mediates their interactions with vascular endothelial cells. FTO deficiency exacerbates retinal inflammation and microvascular dysfunction in diabetic retinas. FTO stabilizes mRNA through an N 6-methyladenosine-YTHDF2–dependent pathway, thereby activating the PI3K/AKT signaling cascade. [ABSTRACT FROM AUTHOR]

We postulated that type 2 diabetes (T2D) predisposes patients to exocrine pancreatic diseases through (epi)genetic mechanisms. We explored the methylome (using MethylationEPIC arrays) of the exocrine pancreas in 141 donors, assessing the impact of T2D. An epigenome-wide association study of T2D identified hypermethylation in an enhancer of the pancreatic lipase–related protein 1 (PNLIPRP1) gene, associated with decreased PNLIPRP1 expression. PNLIPRP1 null variants (found in 191,000 participants in the UK Biobank) were associated with elevated glycemia and LDL cholesterol. Mendelian randomization using 2.5M SNP Omni arrays in 111 donors revealed that T2D was causal of PNLIPRP1 hypermethylation, which in turn was causal of LDL cholesterol. Additional AR42J rat exocrine cell analyses demonstrated that Pnliprp1 knockdown induced acinar-to-ductal metaplasia, a known prepancreatic cancer state, and increased cholesterol levels, reversible with statin. This (epi)genetic study suggests a role for PNLIPRP1 in human metabolism and exocrine pancreatic function, with potential implications for pancreatic diseases. Article Highlights: We performed this study to identify epigenetic changes with type 2 diabetes (T2D) in the pancreas. This study addresses whether T2D induces epigenetic changes that could explain why individuals with T2D are more prone to pancreatic diseases. We found hypermethylation at PNLIPRP1 associated with T2D and identified a role of this gene in cholesterol metabolism. This study has important implications in the prevention of pancreatic diseases, because their molecular mechanisms remain largely unknown. [ABSTRACT FROM AUTHOR]

Endothelial dysfunction plays a crucial role in the progress of diabetic vasculopathy. C1q/tumor necrosis factor-related protein 13 (CTRP13) is a secreted adipokine that can ameliorate atherosclerosis and vascular calcification. However, the role of CTRP13 in regulating endothelial function in diabetes has yet to be explored. In this study, CTRP13 treatment improved endothelium-dependent relaxation in the aortae and mesenteric arteries of both db/dbmice and streptozotocin-injectedmice. CTRP13 supplement also rescued the impaired endothelium-dependent relaxation ex vivo in the db/db mouse aortae and in high glucose (HG)-treated mouse aortae. Additionally, CTRP13 treatment reduced reactive oxygen species overproduction and improved nitric oxide (NO) production and endothelial NO synthase (eNOS) coupling in the aortae of diabetic mice and in HG-treated human umbilical vein endothelial cells. Mechanistically, CTRP13 could increase GTP cyclohydrolase 1 (GCH1) expression and tetrahydrobiopterin (BH4) levels to ameliorate eNOS coupling. More importantly, CTRP13 rescued HG-induced inhibition of protein kinase A (PKA) activity. Increased PKA activity enhanced phosphorylation of the peroxisome proliferator-activated receptor a and its recruitment to the GCH1 promoter, thus activating GCH1 transcription and, ultimately, endothelial relaxation. Together, these results suggest that CTRP13 preserves endothelial function in diabetic mice by regulating GCH1/BH4 axis-dependent eNOS coupling, suggesting the therapeutic potential of CTRP13 against diabetic vasculopathy. [ABSTRACT FROM AUTHOR]

Type 1 diabetes impairs fracture healing. We tested the hypothesis that diabetes affects chondrocytes to impair fracture healing through a mechanism that involves the transcription factor FOXO1. Type 1 diabetes was induced by streptozotocin in mice with FOXO1 deletion in chondrocytes (Col2α1Cre+FOXO1L/L) or littermate controls (Col2α1Cre-FOXO1L/L) and closed femoral fractures induced. Diabetic mice had 77% less cartilage and 30% less bone than normoglycemics evaluated histologically and by micro-computed tomography. Both were reversed with lineage-specific FOXO1 ablation. Diabetic mice had a threefold increase in osteoclasts and a two- to threefold increase in RANKL mRNA or RANKL-expressing chondrocytes compared with normoglycemics. Both parameters were rescued by FOXO1 ablation in chondrocytes. Conditions present in diabetes, high glucose (HG), and increased advanced glycation end products (AGEs) stimulated FOXO1 association with the RANKL promoter in vitro, and overexpression of FOXO1 increased RANKL promoter activity in luciferase reporter assays. HG and AGE stimulated FOXO1 nuclear localization, which was reversed by insulin and inhibitors of TLR4, histone deacetylase, nitric oxide, and reactive oxygen species. The results indicate that chondrocytes play a prominent role in diabetes-impaired fracture healing and that high levels of glucose, AGEs, and tumor necrosis factor-α, which are elevated by diabetes, alter RANKL expression in chondrocytes via FOXO1. [ABSTRACT FROM AUTHOR]

Retinal fibrosis is one of the major features of diabetic retinopathy (DR). Our recent research has shown that Poldip2 can affect early DR through oxidative stress, but whether Poldip2 would regulate retinal fibrosis during DR development is still enigmatic. Here, diabetic Sprague-Dawley (SD) rats were induced with streptozotocin (STZ) and treated with adeno-associated virus serotype 9–polymerase-δ interacting protein 2 (Poldip2) shRNA, while human adult retinal pigment epithelial (ARPE-19) cells were treated with high glucose or Poldip2 siRNA. We identified that in STZ-induced DR rats and ARPE-19 cells treated with high glucose, the expression of Poldip2, transforming growth factor-β1 (TGF-β1), phosphorylated-SMAD3/SMAD3, MMP9, COL-1, FN, and CTGF increased while the expression of cadherin decreased. However, deleting Poldip2 inhibited the TGF-β1/SMAD3 signaling pathway and attenuated the above protein expression in vivo and in vitro. Mechanistically, we found that Poldip2 promotes the activation of SMAD3, facilitates its nuclear translocation through interacting with it, and significantly enhances the expression of fibrosis makers. Collectively, Poldip2 was identified is a novel regulator of DR fibrosis and is expected to become a therapeutic target for PDR. Article Highlights: To investigate the role of polymerase-δ interacting protein 2 (Poldip2) in retinal fibrosis during diabetic retinopathy (DR) development. Does Poldip2 regulate retinal fibrosis in DR, and what are the underlying mechanisms? Poldip2 upregulation in DR correlates with increased expression of fibrotic markers, while Poldip2 deletion inhibits the transforming growth factor-β1/SMAD3 pathway, reducing fibrosis-related protein levels. Poldip2 emerges as a novel regulator of DR fibrosis, suggesting therapeutic potential for treating proliferative diabetic retinopathy. [ABSTRACT FROM AUTHOR]

Diabetic retinopathy is the most common microvascular complication caused by chronic hyperglycemia and is a leading cause of blindness; however, the underlying molecular mechanism has not been clearly elucidated. Therefore, we investigated whether regulation of AMPK and GAPDH by transglutaminase 2 (TGase2) is important for hyperglycemia-induced microvascular leakage in the diabetic retina. In human retinal endothelial cells (HRECs) and diabetic mouse retinas, we found that TGase2, activated by sequential elevation of intracellular Ca2+ and reactive oxygen species (ROS) levels, played an essential role in hyperglycemia-induced vascular leakage. ROS generation and TGsae2 activation were involved in hyperglycemia-induced AMPK dephosphorylation, which resulted in vascular endothelial–cadherin (VE-cadherin) disassembly and increased fluorescein isothiocyanate-dextran extravasation. Furthermore, high glucose–induced TGase2 activation suppressed GAPDH activity, determined by an on-chip activity assay, through inhibition of AMPK, which induced VE-cadherin disassembly and endothelial permeability in HRECs. Overall, our findings suggest that inhibition of AMPK and GAPDH by TGase2 plays a pivotal role in hyperglycemia-induced microvascular leakage in the retinas of diabetic mice. Article Highlights: Reactive oxygen species–mediated activation of transglutaminase 2 (TGase2) inhibited AMPK in the diabetic retina. AMPK inhibition resulted in vascular endothelial–cadherin disassembly and microvascular leakage. TGase2 suppressed GAPDH activity and induced endothelial permeability through AMPK inhibition. Inhibition of AMPK and GAPDH by TGase2 is important for microvascular leakage in the diabetic retina. [ABSTRACT FROM AUTHOR]

Defective fatty acid oxidation (FAO) has been implicated in diabetic kidney disease (DKD), yet little is known about the role of carnitine palmitoyltransferase-1A (CPT1A), a pivotal rate-limiting enzyme of FAO, in the progression of DKD. Here, we investigate whether CPT1A is a reliable therapeutic target for DKD. We first confirmed the downregulation expression of CPT1A in glomeruli from patients with diabetes. We further evaluated the function of CPT1A in diabetic models. Overexpression of CPT1A exhibited protective effects in diabetic conditions, improving albuminuria and glomerular sclerosis as well as mitigating glomerular lipid deposits and podocyte injury in streptozotocin-induced diabetic mice. Mechanistically, CPT1A not only fostered lipid consumption via fatty acid metabolism pathways, thereby reducing lipotoxicity, but also anchored Bcl2 to the mitochondrial membrane, thence preventing cytochrome C release and inhibiting the mitochondrial apoptotic process. Furthermore, a novel transcription factor of CPT1A, FOXA1, was identified. We elucidate the crucial role of CPT1A in mitigating podocyte injury and the progression of DKD, indicating that targeting CPT1A may be a promising avenue for DKD treatment. Article Highlights: Fatty acid oxidation has been a critical contributor to podocyte injury and pathological changes in diabetic kidney disease. However, the role of carnitine palmitoyltransferase-1A (CPT1A), which is the initial key rate-limiting enzyme of mitochondrial fatty acid oxidation in diabetic kidney disease, remains largely unknown. We investigate whether CPT1A modulates the progression of diabetic kidney disease. CPT1A ameliorated albuminuria and glomerular sclerosis, and mitigated glomerular lipid deposits and podocyte injury in streptozotocin-induced diabetic mice. Our study reveals a promising avenue for diabetic kidney disease prevention and clarifies a new regulatory mechanism for CPT1A in podocytes. [ABSTRACT FROM AUTHOR]

Increasing evidence implicates chronic inflammation as the main pathological cause of diabetic nephropathy (DN). Exploration of key targets in the inflammatory pathway may provide new treatment options for DN. We aimed to investigate the role of Src homology 2–containing protein tyrosine phosphatase 2 (SHP2) in macrophages and its association with DN. The upregulated phosphorylation of SHP2 was detected in macrophages in both patients with diabetes and in a mouse model. Using macrophage-specific SHP2-knockout (SHP2-MKO) mice and SHP2fl/fl mice injected with streptozotocin (STZ), we showed that SHP2-MKO significantly attenuated renal dysfunction, collagen deposition, fibrosis, and inflammatory response in mice with STZ-induced diabetes. RNA-sequencing analysis using primary mouse peritoneal macrophages (MPMs) showed that SHP2 deletion mainly affected mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) signaling pathways as well as MAPK/NF-κB–dependent inflammatory cytokine release in MPMs. Further study indicated that SHP2-deficient macrophages failed to release cytokines that induce phenotypic transition and fibrosis in renal cells. Administration with a pharmacological SHP2 inhibitor, SHP099, remarkably protected kidneys in both type 1 and type 2 diabetic mice. In conclusion, these results identify macrophage SHP2 as a new accelerator of DN and suggest that SHP2 inhibition may be a therapeutic option for patients with DN. Article Highlights: Phosphorylated Src homology 2–containing protein tyrosine phosphatase 2 (SHP2) levels in macrophages are increased in both patients and mice with diabetic neuropathy (DN). Macrophage SHP2 knockout prevents renal inflammation and DN in diabetic mice. SHP2 deletion suppresses the activation of mitogen-activated protein kinase and nuclear factor-κB signaling pathways, thereby inhibiting inflammation in macrophages. Pharmacological inhibition of SHP2 protects against DN in both type 1 and type 2 diabetic mice. [ABSTRACT FROM AUTHOR]

Hydrogen sulfide (H2S) has been shown to have powerful antioxidative and anti-inflammatory properties that can regulate multiple cardiovascular functions. However, its precise role in diabetes-accelerated atherosclerosis remains unclear. We report here that H2S reduced aortic atherosclerotic plaque formation with reduction in superoxide (O2 (-)) generation and the adhesion molecules in streptozotocin (STZ)-induced LDLr(-/-) mice but not in LDLr(-/-)Nrf2(-/-) mice. In vitro, H2S inhibited foam cell formation, decreased O2 (-) generation, and increased nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation and consequently heme oxygenase 1 (HO-1) expression upregulation in high glucose (HG) plus oxidized LDL (ox-LDL)-treated primary peritoneal macrophages from wild-type but not Nrf2(-/-) mice. H2S also decreased O2 (-) and adhesion molecule levels and increased Nrf2 nuclear translocation and HO-1 expression, which were suppressed by Nrf2 knockdown in HG/ox-LDL-treated endothelial cells. H2S increased S-sulfhydration of Keap1, induced Nrf2 dissociation from Keap1, enhanced Nrf2 nuclear translocation, and inhibited O2 (-) generation, which were abrogated after Keap1 mutated at Cys151, but not Cys273, in endothelial cells. Collectively, H2S attenuates diabetes-accelerated atherosclerosis, which may be related to inhibition of oxidative stress via Keap1 sulfhydrylation at Cys151 to activate Nrf2 signaling. This may provide a novel therapeutic target to prevent atherosclerosis in the context of diabetes. [ABSTRACT FROM AUTHOR]

Epidemiologic studies suggest that hepatocellular carcinoma (HCC) has a strong relationship with diabetes. However, the underlying molecular mechanisms still remain unclear. Here, we demonstrated that high glucose (HG), one of the main characteristics of diabetes, was capable of accelerating tumorigenesis in HCC cells. Advanced glycosylation end product-specific receptor (AGER) was identified as a stimulator during this process. Mechanistically, AGER activated a hexosamine biosynthetic pathway, leading to enhanced O-GlcNAcylation of target proteins. Notably, AGER was capable of increasing activity and stability of proto-oncoprotein c-Jun via O-GlcNAcylation of this protein at Ser73. Interestingly, c-Jun can conversely enhance AGER transcription. Thereby, a positive autoregulatory feedback loop that stimulates diabetic HCC was established. Finally, we found that AG490, an inhibitor of Janus kinase, has the ability to impair AGER expression and its functions in HCC cells. In conclusion, AGER and its functions to stimulate O-GlcNAcylation are important during liver tumorigenesis, when high blood glucose levels are inadequately controlled. [ABSTRACT FROM AUTHOR]

Vascular inflammation is known to cause degeneration of retinal capillaries in early diabetic retinopathy (DR), a major microvascular complication of diabetes. Past studies investigating these diabetes-induced retinal vascular abnormalities have focused primarily on the role of molecular or biochemical cues. Here we show that retinal vascular inflammation and degeneration in diabetes are also mechanically regulated by the increase in retinal vascular stiffness caused by overexpression of the collagen–cross-linking enzyme lysyl oxidase (LOX). Treatment of diabetic mice with LOX inhibitor β-aminopropionitrile (BAPN) prevented the increase in retinal capillary stiffness, vascular intracellular adhesion molecule-1 overexpression, and leukostasis. Consistent with these anti-inflammatory effects, BAPN treatment of diabetic mice blocked the upregulation of proapoptotic caspase-3 in retinal vessels, which concomitantly reduced retinal capillary degeneration, pericyte ghost formation, and the diabetes-induced loss of contrast sensitivity in these mice. Finally, our in vitro studies indicate that retinal capillary stiffening is sufficient to increase the adhesiveness and neutrophil elastase-induced death of retinal endothelial cells. By uncovering a link between LOX-dependent capillary stiffening and the development of retinal vascular and functional defects in diabetes, these findings offer a new insight into DR pathogenesis that has important translational potential. Article Highlights: The objective of this study was to determine whether retinal vascular inflammation and degeneration associated with early diabetic retinopathy are mechanically regulated by the increased stiffness of retinal capillaries. We provide the first direct evidence of retinal capillary stiffening in diabetes that is dependent on lysyl oxidase (LOX), promotes retinal vascular inflammation, and causes capillary degeneration by increasing retinal endothelial susceptibility to neutrophil elastase. We also show that pharmacological inhibition of LOX prevents the diabetes-induced loss of contrast sensitivity. These findings implicate LOX and capillary stiffening as new anti-inflammatory targets for the treatment of retinal vascular and functional defects associated with early diabetic retinopathy. [ABSTRACT FROM AUTHOR]

Hyperglycemia-induced inflammation and apoptosis have important roles in the pathogenesis of diabetic cardiomyopathy. We recently found that a novel curcumin derivative, C66, is able to reduce the high glucose (HG)-induced inflammatory response. This study was designed to investigate the protective effects on diabetic cardio-myopathy and its underlying mechanisms. Pretreatment with C66 significantly reduced HG-induced overex-pression of inflammatory cytokines via inactivation of nuclear factor-κB in both H9c2 cells and neonatal cardiomyocytes. Furthermore, we showed that the inhibition of Jun NH2-terminal kinase (JNK) phosphorylation contributed to the protection of C66 from inflammation and cell apoptosis, which was validated by the use of SP600125 and dominant-negative JNK. The molecular docking and kinase activity assay confirmed direct binding of C66 to and inhibition of JNK. In mice with type 1 diabetes, the administration of C66 or SP600125 at 5 mg/kg significantly decreased the levels of plasma and cardiac tumor necrosis factor-α, accompanied by decreasing cardiac apoptosis, and, finally, improved histological abnormalities, fibrosis, and cardiac dysfunction without affecting hyperglycemia. Thus, this work demonstrated the therapeutic potential of the JNK-targeting compound C66 for the treatment of diabetic cardiomyopathy. Importantly, we indicated a critical role of JNK in diabetic heart injury, and suggested that JNK inhibition may be a feasible strategy for treating diabetic cardiomyopathy. [ABSTRACT FROM AUTHOR]

Rap1b ameliorates high glucose (HG)-induced mitochondrial dysfunction in tubular cells. However, its role and precise mechanism in diabetic nephropathy (DN) in vivo remain unclear. We hypothesize that Rap1 plays a protective role in tubular damage of DN by modulating primarily the mitochondria-derived oxidative stress. The role and precise mechanisms of Rap1b on mitochondrial dysfunction and of tubular cells in DN were examined in rats with streptozotocin (STZ)-induced diabetes that have Rap1b gene transfer using an ultrasound microbubble-mediated technique as well as in renal proximal epithelial tubular cell line (HK-2) exposed to HG ambiance. The results showed that Rap1b expression decreased significantly in tubules of renal biopsies from patients with DN. Overexpression of a constitutively active Rap1b G12V notably ameliorated renal tubular mitochondrial dysfunction, oxidative stress, and apoptosis in the kidneys of STZ-induced rats, which was accompanied with increased expression of transcription factor C/EBP-β and PGC-1α. Furthermore, Rap1b G12V also decreased phosphorylation of Drp-1, a key mitochondrial fission protein, while boosting the expression of genes related to mitochondrial biogenesis and antioxidants in HK-2 cells induced by HG. These effects were imitated by transfection with C/EBP-β or PGC-1α short interfering RNA. In addition, Rap1b could modulate C/EBP-β binding to the endogenous PGC-1α promoter and the interaction between PGC-1α and catalase or mitochondrial superoxide dismutase, indicating that Rap1b ameliorates tubular injury and slows the progression of DN by modulation of mitochondrial dysfunction via C/EBP-β-PGC-1α signaling. [ABSTRACT FROM AUTHOR]

Podocyte apoptosis is a critical mechanism for excessive loss of urinary albumin that eventuates in kidney fibrosis. Pharmacological doses of the mammalian target of rapamycin (mTOR) inhibitor rapamycin reduce albuminuria in diabetes. We explored the hypothesis that mTOR mediates podocyte injury in diabetes. High glucose (HG) induces apoptosis of podocytes, inhibits AMP-activated protein kinase (AMPK) activation, inactivates tuberin, and activates mTOR. HG also increases the levels of Nox4 and Nox1 and NADPH oxidase activity. Inhibition of mTOR by low-dose rapamycin decreases HG-induced Nox4 and Nox1, NADPH oxidase activity, and podocyte apoptosis. Inhibition of mTOR had no effect on AMPK or tuberin phosphorylation, indicating that mTOR is downstream of these signaling molecules. In isolated glomeruli of OVE26 mice, there is a similar decrease in the activation of AMPK and tuberin and activation of mTOR with increase in Nox4 and NADPH oxidase activity. Inhibition of mTOR by a small dose of rapamycin reduces podocyte apoptosis and attenuates glomerular injury and albuminuria. Our data provide evidence for a novel function of mTOR in Nox4-derived reactive oxygen species generation and podocyte apoptosis that contributes to urinary albumin excretion in type 1 diabetes. Thus, mTOR and/or NADPH oxidase inhibition may represent a therapeutic modality of diabetic kidney disease. [ABSTRACT FROM AUTHOR]

Protein kinase C (PKC) β2 is preferably overexpressed in the diabetic myocardium, which induces cardiomyocyte hypertrophy and contributes to diabetic cardiomyopathy, but the underlying mechanisms are incompletely understood. Caveolae are critical in signal transduction of PKC isoforms in cardiomyocytes. Caveolin (Cav)-3, the cardiomyocyte-specific caveolar structural protein isoform, is decreased in the diabetic heart. The current study determined whether PKCβ2 activation affects caveolae and Cav-3 expression. Immunoprecipitation and immunofluorescence analysis revealed that high glucose (HG) increased the association and colocaliza-tion of PKCβ2 and Cav-3 in isolated cardiomyocytes. Disruption of caveolae by methyl-β-cyclodextrin or Cav-3 small interfering (si)RNA transfection prevented HG-induced PKCβ2 phosphorylation. Inhibition of PKCβ2 activation by compound CGP53353 or knockdown of PKCβ2 expression via siRNA attenuated the reductions of Cav-3 expression and Akt/endothelial nitric oxide synthase (eNOS) phosphorylation in cardiomyocytes exposed to HG. LY333531 treatment (for a duration of 4 weeks) prevented excessive PKCβ2 activation and attenuated cardiac diastolic dysfunction in rats with streptozotocin-induced diabetes. LY333531 suppressed the decreased expression of myocardial NO, Cav-3, phosphorylated (p)-Akt, and p-eNOS and also mitigated the augmentation of O2-, nitrotyrosine, Cav-1, and iNOS expression. hi conclusion, hyperglycemia-induced PKCβ2 activation requires caveolae and is associated with reduced Cav-3 expression in the diabetic heart. Prevention of excessive PKCβ2 activation attenuated cardiac diastolic dysfunction by restoring Cav-3 expression and subsequently rescuing Akt/eNOS/NO signaling. [ABSTRACT FROM AUTHOR]

OBJECTIVE--Adverse effects of hypercaloric, high-fructose diets on insulin sensitivity and lipids in human subjects have been shown repeatedly. The implications of fructose in amounts close to usual daily consumption, however, have not been well studied. This study assessed the effect of moderate amounts of fructose and sucrose compared with glucose on glucose and lipid metabolism. RESEARCH DESIGN AND METHODS--Nine healthy, normal-weight male volunteers (aged 21-25 years) were studied in this double-blind, randomized, cross-over trial. All subjects consumed four different sweetened beverages (600 mL/day) for 3 weeks each: medium fructose (MF) at 40 g/day, and high fructose (HF), high glucose (HG), and high sucrose (HS) each at 80 g/day. Euglycemic-hyperinsulinemic clamps with [6,6]-²H2 glucose labeling were used to measure endogenous glucose production. Lipid profile, glucose, and insulin were measured in fasting samples. RESULTS--Hepatic suppression of glucose production during the clamp was significantly lower after HF (59.4 ± 11.0%) than HG (70.3 ± 10.5%, P < 0.05), whereas fasting glucose, insulin, and C-peptide did not differ between the interventions. Compared with HG, LDL cholesterol and total cholesterol were significantly higher after MF, HF, and HS, and free fatty acids were significantly increased after MF, but not after the two other interventions (P < 0.05). Subjects' energy intake during the interventions did not differ significantly from baseline intake. CONCLUSIONS--This study clearly shows that moderate amounts of fructose and sucrose significantly alter hepatic insulin sensitivity and lipid metabolism compared with similar amounts of glucose. [ABSTRACT FROM AUTHOR]

In type 2 diabetes, hyperglycemia and increased sympathetic drive may alter mitochondria energetic/redox properties, decreasing the organelle's functionality. These perturbations may prompt or sustain basal low-cardiac performance and limited exercise capacity. Yet the precise steps involved in this mitochondrial failure remain elusive. Here, we have identified dysfunctional mitochondrial respiration with substrates of complex I, II, and IV and lowered thioredoxin-2/glutathione (GSH) pools as the main processes accounting for impaired state 4→3 energetic transition shown by mitochondria from hearts of type 2 diabetic db/db mice upon challenge with high glucose (HG) and the b-agonist isoproterenol (ISO). By mimicking clinically relevant conditions in type 2 diabetic patients, this regimen triggers a major overflow of reactive oxygen species (ROS) from mitochondria that directly perturbs cardiac electro-contraction coupling, ultimately leading to heart dysfunction. Exogenous GSH or, even more so, the fatty acid palmitate rescues basal and β-stimulated function in db/db myocyte/ heart preparations exposed to HG/ISO. This occurs because both interventions provide the reducing equivalents necessary to counter mitochondrial ROS outburst and energetic failure. Thus, in the presence of poor glycemic control, the diabetic patient's inability to cope with increased cardiac work demand largely stems from mitochondrial redox/energetic disarrangements that mutually influence each other, leading to myocyte or whole-heart mechanical dysfunction. [ABSTRACT FROM AUTHOR]

The current study investigated the potential of green tea (GT) to improve uncoupling of endothelial nitric oxide synthase (eNOS) in diabetic conditions. In rats with streptozotocin-induced diabetes, nitric oxide (NO) bioavailability was reduced by uncoupling eNOS, characterized by a reduction in tetrahydrobiopterin (BH4) levels and a decrease in the eNOS dimer-to-monomer ratio. GT treatment ameliorated these abnormalities. Moreover, immortalized human mesangial cells (ihMCs) exposed to high glucose (HG) levels exhibited a rise in reactive oxygen species (ROS) and a decline in NO levels, which were reversed with GT. BH4 and the activity of guanosine triphosphate cyclohydrolase I decreased in ihMCs exposed to HG and was normalized by GT. Exogenous administration of BH4 in ihMCs reversed the HG-induced rise in ROS and the decline in NO production. However, coadministration of GT with BH4 did not result in a further reduction in ROS production, suggesting that reduced ROS with GT was indeed secondary to uncoupled eNOS. In summary, GT reversed the diabetes- induced reduction of BH4 levels, ameliorating uncoupling eNOS, and thus increasing NO bioavailability and reducing oxidative stress, two abnormalities that are involved in the pathogenesis of diabetic nephropathy. [ABSTRACT FROM AUTHOR]

The objectives of this study were to investigate how diabetes mellitus (DM) influences responsiveness of retinal neovessels to lysophosphatidic acid (LPA) and to elucidate the underlying mechanism. To this end, we used an ex vivo assay in which neovessels sprouted from retinal explants (isolated from either control or DM mice) when cultured between two layers of collagen and in the presence of vascular endothelial growth factor-A. While DM had no effect on the formation of neovessels, it prevented LPA-induced regression. High-glucose (HG) treatment of retinal explants mimicked the DM phenotype. Similarly, primary retinal endothelial cells (RECs), which were subjected to HG treatment, organized into tubes that were resistant to LPA. HG caused LPA resistance within RECs by elevating ROS, which activated Src-family kinases that stimulated the extracellular signal--related kinase (Erk) pathway, which antagonized LPA-mediated signaling events that were required for regression. This ROS/Src/Erk pathway mechanism appeared to be the same route by which DM induced LPA resistance of retinal neovessels. We conclude that DM/HG reprograms signaling pathways in RECs to induce a state of LPA resistance. [ABSTRACT FROM AUTHOR]

Clinical studies suggest metabolic memory to hyperglycema. We tested whether diabetes leads to persistent systematic in vitro gene expression alterations in patients with type 1 diabetes (T1D) compared with their monozygotic, nondiabetic twins. Microarray gene expression was determined in skin fibroblasts (SFs) of five twin pairs cultured in high glucose (HG) for ~6 weeks. The Exploratory Visual Analysis System tested group differences in gene expression levels within KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways. An overabundance of differentially expressed genes was found in eight pathways: arachidonic acid metabolism (P = 0.003849), transforming growth factor-β signaling (P = 0.009167), glutathione metabolism (P = 0.01281), glycosylphosphatidylinositol anchor (P = 0.01949), adherens junction (P = 0.03134), dorsal-ventral axis formation (P = 0.03695), proteasome (P = 0.04327), and complement and coagulation cascade (P = 0.04666). Several genes involved in epigenetic mechanisms were also differentially expressed. All differentially expressed pathways and all the epigenetically relevant differentially expressed genes have previously been related to HG in vitro or to diabetes and its complications in animal and human studies. However, this is the first in vitro study demonstrating diabetes-relevant gene expression differences between TID-discordant identical twins. These SF gene expression differences, persistent despite the HG in vitro conditions, likely reflect "metabolic memory", and discordant identical twins thus represent an excellent model for studying diabetic epigenetic processes in humans. [ABSTRACT FROM AUTHOR]

OBJECTIVE--In diabetes, retinal vascular basement membrane (BM) undergoes significant thickening and compromises vessel function including increased vascular permeability, a prominent lesion of early diabetic retinopathy. In this study we determined whether altered expression and activity of lysyl oxidase (LOX), a cross-linking enzyme, may compromise vascular basement membrane functional integrity under high-glucose (HG) conditions. RESEARCH DESIGN AND METHODS--Rat retinal endothelial cells (RRECs) grown in normal (5 mmol/l) or HG (30 mmol/l glucose) medium for 7 days were assessed for expression of LOX and proLOX by Western blot analysis and LOX enzyme activity. To determine whether HG alters cellular distribution patterns of LOX and proLOX, immunostaining with respective antibodies was performed. Similarly, cells grown in normal or HG medium were subjected to both LOX inhibition with β-aminopropionitrile (BAPN) and by small interfering RNA knockdown, and respectively examined for cell monolayer permeability. Additionally, retinas of streptozotocin (STZ)-induced diabetic rats were analyzed to determine if diabetes altered LOX expression. RESULTS--Western blot analysis revealed significantly increased LOX and proLOX expression in cells grown in HG medium compared with those grown in normal medium. The increased LOX level was strikingly similar to LOX upregulation in the diabetic retinas. In cells grown in HG medium, LOX activity and cell monolayer permeability was significantly increased, as were LOX and proLOX immunostaining. Small interfering RNA- or BAPN-induced-specific blockage of LOX expression or activity, respectively, reduced cell monolayer permeability. CONCLUSIONS--HG-induced increased LOX expression and activity compromises barrier functional integrity, a prominent lesion of diabetic retinopathy. Diabetes 59:3159-3166, 2010 [ABSTRACT FROM AUTHOR]

OBJECTIVE--Disturbances in podocytes are typically associated with marked proteinuria, a hallmark of diabetic nephropathy. This study was conducted to investigate modulation of Notch-1 signaling in high glucose (HG)-stressed human podocytes and in a diabetic animal model. RESEARCH DESIGN AND METHODS--Expression of the Notch signaling components was examined in HG-treated podocytes, human embryonic kidney cells (HEK293), and kidneys from diabetic animals by RT-qPCR, Western blot analysis, and immunohistochemical staining. The association between the Notch signaling, VEGF expression, and podocyte integrity was evaluated. RESULTS--Notch-1 signaling was significantly activated in HG-cultured human podocytes and HEK293 cells and kidneys from diabetic animals. HG also augmented VEGF expression, decreasing nephrin expression and podocyte number--a critical event for the development of proteinuria in diabetic nephropathy. After use of pharmacological modulators or specific shRNA knockdown strategies, inhibition of Notch-1 signaling significantly abrogated VEGF activation and nephrin repression in HG-stressed cells and ameliorated proteinuria in the diabetic kidney. CONCLUSIONS--Our findings suggest that upregulation of Notch-1 signaling in HG-treated renal podocytes induces VEGF expression and subsequent nephrin repression and apoptosis. Modulation of Notch-1 signaling may hold promise as a novel therapeutic strategy for the treatment of diabetic nephropathy. Diabetes 59:1915-1925, 2010 [ABSTRACT FROM AUTHOR]

Coronary microvessels generate reactive oxygen species in response to high glucose (HG), resulting in vasodilator defects involving an impaired function of vascular K(+) channels. Inhibition of voltage-gated K(+) (K(v)) channels by peroxynitrite (ONOO(-)), formed by the interaction of superoxide and nitric oxide, may contribute to impaired dilation. The present study investigated whether HG induces ONOO(-) formation to mediate nitration and impairment of K(v) channels in rat small coronary arteries (RSCAs). Exposure to ONOO(-) reduced the dilator influence of K(v) channels in RSCAs. Patch-clamp studies revealed that ONOO(-) diminished whole-cell and unitary K(v) currents attributable to the K(v)1 gene family in smooth muscle cells. Subsequently, immunohistochemically detected enhancement of nitrotyrosine residues in RSCAs that were cultured in HG (23 mmol/l) compared with normal glucose (5.5 mmol/l) for 24 h correlated with the nitration of K(v)1.2 channel alpha-subunits. HG-induced nitrotyrosine formation was partially reversed by scavenging ONOO(-). Finally, RSCAs that were exposed to HG for 24 h showed a loss of K(v) channel dilator influence that also was partially restored by the ONOO(-) scavengers urate and ebselen. We conclude that ONOO(-) generated by HG impairs K(v) channel function in coronary microvessels, possibly by nitrating tyrosine residues in the pore-forming region of the K(v) channel protein. [ABSTRACT FROM AUTHOR]

Monocyte activation and adhesion to the endothelium play important roles in inflammatory and cardiovascular diseases. These processes are further aggravated by hyperglycemia, leading to cardiovascular complications in diabetes. We have previously shown that high glucose (HG) treatment activates monocytes and induces the expression of tumor necrosis factor (TNF)-alpha via oxidant stress and nuclear factor-kB transcription factor. To determine the effects of HG on the expression of other inflammatory genes, in the present study, HG-induced gene profiling was performed in THP-1 monocytes using cytokine gene arrays containing 375 known genes. HG treatment upregulated the expression of 41 genes and downregulated 15 genes that included chemokines, cytokines, chemokines receptors, adhesion molecules, and integrins. RT-PCR analysis further confirmed that HG significantly increased the expression of monocyte chemoattractant protein-1 (MCP-1), TNF-alpha, beta(2)-integrin, interleukin-1beta, and others. HG treatment increased transcription of the MCP-1 gene, MCP-1 protein levels, and adhesion of THP-1 cells to endothelial cells. HG-induced MCP-1 mRNA expression and monocyte adhesion were blocked by specific inhibitors of oxidant stress, protein kinase C, ERK1/2, and p38 mitogen-activated protein kinases. These results show for the first time that multiple inflammatory cytokines and chemokines relevant to the pathogenesis of diabetes complications are induced by HG via key signaling pathways. [ABSTRACT FROM AUTHOR]

High glucose (HG) causes glomerular mesangial cell (GMC) growth, production of transforming growth factor (TGF)-beta, and increased synthesis of matrix proteins such as fibronectin, contributing to diabetic nephropathy. We recently found that exposure of cells to HG also activates the growth-promoting enzyme janus kinase 2 (JAK2) and its latent signal transducers and activators of transcription (STAT) transcription factors (STAT1, STAT3, and STAT5). Our purpose was to determine the effect that inhibition of JAK2 and these STAT transcription factors has on the HG-induced increase in TGF-beta and fibronectin synthesis in GMC. Exposure of GMC to 25 mmol/l glucose caused the activation of JAK2, STAT1, STAT3, and STAT5 plus an increase in TGF-beta and fibronectin synthesis, as compared with 5.5 mmol/l glucose. This HG-induced increase in synthesis of TGF-beta and fibronectin was prevented by concomitant incubation with AG-490, a specific JAK2 inhibitor. The HG-induced JAK2, STAT1, and STAT3 tyrosine phosphorylations in GMC were also abolished by AG-490. Preincubation of GMC cultured in 25 mmol/l glucose with a specific JAK2 or STAT1 antisense oligonucleotide also prevented both TGF-beta and fibronectin synthesis. These results provide direct evidence for linkages between JAK2, STAT1, and the glucose-induced overproduction of TGF-beta and fibronectin in GMC. [ABSTRACT FROM AUTHOR]

We examined the effect of hypoxia on proliferation and osteopontin (OPN) expression in cultured rat aortic vascular smooth muscle (VSM) cells. In addition, we determined whether hypoxia-induced increases in OPN and cell proliferation are altered under hyperglycemic conditions. Quiescent cultures of VSM cells were exposed to hypoxia (3% O(2)) or normoxia (18% O(2)) in a serum-free medium, and cell proliferation as well as the expression of OPN was assessed. Cells exposed to hypoxia for 24 h exhibited a significant increase in [(3)H]thymidine incorporation followed by a significant increase in cell number at 48 h in comparison with respective normoxic controls. Exposure to hypoxia produced significant increases in OPN protein and mRNA expression at 2 h followed by a gradual decline at 6 and 12 h, with subsequent significant increases at 24 h. Neutralizing antibodies to either OPN or its receptor beta3 integrin but not neutralizing antibodies to beta5 integrin prevented the hypoxia-induced increase in [(3)H]thymidine incorporation. Inhibitors of protein kinase C (PKC) and p38 mitogen-activated protein (MAP) kinase also reduced the hypoxia-induced stimulation of proliferation and OPN synthesis. Exposure to high-glucose (HG) (25 mmol/l) medium under normoxic conditions also resulted in significant increases in OPN protein and mRNA levels as well as the proliferation of VSM cells. Under hypoxic conditions, HG further stimulated OPN synthesis and cell proliferation in an additive fashion. In conclusion, hypoxia-induced proliferation of cultured VSM cells is mediated by the stimulation of OPN synthesis involving PKC and p38 MAP kinase. In addition, hypoxia also enhances the effect of HG conditions on both OPN and proliferation of cultured VSM cells, which may have important implications in the development of diabetic atherosclerosis associated with arterial wall hypoxia. [ABSTRACT FROM AUTHOR]

Nonenzymatic glycation has been implicated in the pathogenesis of the dysregulated tissue remodeling that characterizes diabetic glomerulopathy, via the formation of advanced glycation end products (AGEs) and their binding to cell surface receptors. Several AGE-binding proteins have been identified so far, including p60, p90, and the adhesive and growth-regulating lectin galectin-3 (Gal-3), the components of the so-called AGE-receptor complex. This study aimed to evaluate the mesangial expression of the AGE-receptor complex and its modulation by the diabetic milieu, both in vivo, in non-diabetic versus streptozotocin-induced diabetic rats, and in vitro, in mesangial cells exposed to either normal glucose (NG) levels (5.5 mmol/l), as compared with high glucose (HG) levels (30 mmol/l) and iso-osmolar mannitol (M), or to native bovine serum albumin (BSA), as compared with glycated BSA with AGE formation (BSA-AGE) and glycated BSA in which AGE formation was prevented by aminoguanidine (BSA-AM). In vivo, Gal-3 protein and mRNA were not detectable in glomeruli from nondiabetic rats until 12 months after initiating the study. On the contrary, in diabetic rats, Gal-3 expression was observed at 2 months of disease duration, and it increased thereafter. Both p60 and p90 immunoreactivities were observed at the glomerular level with slightly increased expression of p90, but not p60, in diabetic versus nondiabetic animals. In vitro, Gal-3 was not detectable in mesangial cells cultured in NG (although it became evident after a certain number of passages in culture), whereas Gal-3 was detectable in cells grown on BSA. Prolonged exposure (2-4 weeks) of mesangial cells to HG but not to M, as well as growing cells on BSA-AGE and, to a lesser extent, BSA-AM, induced or significantly increased the expression of Gal-3, both protein (up to 2.65-fold) and mRNA (up to 3.10-fold) and its secretion in the medium (by approximately 50%). Both p60 and p90 were demonstrated in mesangial cells under NG conditions, and the expression of p90, but not p60, was upregulated by approximately 20% by HG or BSA-AGE. These results indicate that 1) under basal conditions, Gal-3, unlike p90 and p60, is not detectable in the mesangium but becomes expressed with aging and 2) the diabetic milieu induces or upregulates Gal-3 production, whereas it increases only slightly the expression of p90, but not p60. Gal-3 expression or overexpression may modulate the AGE-receptor-mediated events by modifying the function of the AGE-receptor complex. Additionally, it may exert direct effects on tissue remodeling by virtue of its adhesive and growth-regulating properties. [ABSTRACT FROM AUTHOR]

Wound healing is a complex, highly regulated process and is substantially disrupted by diabetes. We show here that human wound healing induces specific epigenetic changes that are exacerbated by diabetes in an animal model. We identified epigenetic changes and gene expression alterations that significantly reduce reepithelialization of skin and mucosal wounds in an in vivo model of diabetes, which were dramatically rescued in vivo by blocking these changes. We demonstrate that high glucose altered FOXO1–matrix metallopeptidase 9 (MMP9) promoter interactions through increased demethylation and reduced methylation of DNA at FOXO1 binding sites and also by promoting permissive histone-3 methylation. Mechanistically, high glucose promotes interaction between FOXO1 and RNA polymerase-II (Pol-II) to produce high expression of MMP9 that limits keratinocyte migration. The negative impact of diabetes on reepithelialization in vivo was blocked by specific DNA demethylase inhibitors in vivo and by blocking permissive histone-3 methylation, which rescues FOXO1-impaired keratinocyte migration. These studies point to novel treatment strategies for delayed wound healing in individuals with diabetes. They also indicate that FOXO1 activity can be altered by diabetes through epigenetic changes that may explain other diabetic complications linked to changes in diabetes-altered FOXO1-DNA interactions. Article Highlights: FOXO1 expression in keratinocytes is needed for normal wound healing. In contrast, FOXO1 expression interferes with the closure of diabetic wounds. Using matrix metallopeptidase 9 as a model system, we found that high glucose significantly increased FOXO1-matrix metallopeptidase 9 interactions via increased DNA demethylation, reduced DNA methylation, and increased permissive histone-3 methylation in vitro. Inhibitors of DNA demethylation and permissive histone-3 methylation improved the migration of keratinocytes exposed to high glucose in vitro and the closure of diabetic skin and mucosal wounds in vivo. Inhibition of epigenetic enzymes that alter FOXO1-induced gene expression dramatically improves diabetic healing and may apply to other conditions where FOXO1 has a detrimental role in diabetic complications. [ABSTRACT FROM AUTHOR]

Type 2 diabetes is a progressive disorder denoted by hyperglycemia and impaired insulin secretion. Although a decrease in β-cell function and mass is a well-known trigger for diabetes, the comprehensive mechanism is still unidentified. Here, we performed single-cell RNA sequencing of pancreatic islets from prediabetic and diabetic db/db mice, an animal model of type 2 diabetes. We discovered a diabetes-specific transcriptome landscape of endocrine and nonendocrine cell types with subpopulations of β- and α-cells. We recognized a new prediabetic gene, Anxa10, that was induced by and regulated Ca2+ influx from metabolic stresses. Anxa10-overexpressed β-cells displayed suppression of glucose-stimulated intracellular Ca2+ elevation and potassium-induced insulin secretion. Pseudotime analysis of β-cells predicted that this Ca2+-surge responder cluster would proceed to mitochondria dysfunction and endoplasmic reticulum stress. Other trajectories comprised dedifferentiation and transdifferentiation, emphasizing acinar-like cells in diabetic islets. Altogether, our data provide a new insight into Ca2+ allostasis and β-cell failure processes. Article Highlights: The transcriptome of single-islet cells from healthy, prediabetic, and diabetic mice was studied. Distinct β-cell heterogeneity and islet cell-cell network in prediabetes and diabetes were found. A new prediabetic β-cell marker, Anxa10, regulates intracellular Ca2+ and insulin secretion. Diabetes triggers β-cell to acinar cell transdifferentiation. [ABSTRACT FROM AUTHOR]

Hemopexin (HPX) is overexpressed in the retina of patients with diabetes and induces the breakdown of the blood-retinal barrier in vitro. The aim of this study was to evaluate whether HPX blockade by specific antibodies (aHPX) could avoid vascular leakage in vivo and microvascular angiogenesis in vitro and ex vivo. For this purpose, the effect of intravitreal (IVT) injections of aHPX on vascular leakage was evaluated in db/db mice and rats with streptozotocin-induced diabetes using the Evans Blue method. Retinal neurodegeneration and inflammation were also evaluated. The antiangiogenic effect of aHPX on human retinal endothelial cells (HRECs) was tested by scratch wound healing and tube formation using standardized methods, as well as by choroidal sprouting assays from retinal explants obtained in rats. We found that IVT injection of aHPX significantly reduced vascular leakage, retinal neurodegeneration, and inflammation. In addition, treatment with aHPX significantly reduced HREC migration and tube formation induced by high glucose concentration and suppressed choroidal sprouting even after vascular endothelial growth factor stimulation, with this effect being higher than obtained with bevacizumab. The antipermeability and antiangiogenic effects of IVT injection of aHPX suggest the blockade or inhibition of HPX as a new strategy for the treatment of advanced stages of diabetic retinopathy. Article Highlights: Hemopexin (HPX) is the best-characterized permeability factor in steroid-sensitive nephrotic syndrome. We have previously reported that HPX is overexpressed in the retina of patients with diabetes and induces the breakdown of the blood-retinal barrier in vitro. Here, we report that intravitreal injection of anti-HPX antibodies significantly reduces vascular leakage, retinal neurodegeneration, and inflammation in diabetic murine models and that the immunoneutralization of HPX exerts a significant antiangiogenic effect in vitro and in retinal explants. The blockade of HPX can be considered as a new therapy for advanced stages of diabetic retinopathy. [ABSTRACT FROM AUTHOR]

We have recently demonstrated that adult rat ventricular myocytes maintained in a high glucose (HG) culture medium exhibit abnormalities in excitation-contraction coupling similar to myocytes from diabetic rats. Metformin, an insulin-sensitizing biguanide, enhances peripheral insulin action and lowers blood pressure in hyperinsulinemic animals, but its direct impact on cardiac function is not fully understood. To examine the role of metformin on HG-induced cardiac dysfunction at the cellular level, normal adult ventricular myocytes were cultured for 1 day in a serum-free insulin-containing medium with either normal glucose (5.5 mmol/l glucose) or HG (25.5 mmol/l glucose) in the presence or absence of metformin or the sulfonylurea glyburide. Mechanical properties were evaluated using a high-speed video-edge detection system, and intracellular Ca2+ transients were recorded in fura-2-loaded myocytes. As previously reported, culturing myocytes in HG depresses peak shortening, prolongs time to 90% relengthening, and slows Ca2+ transient decay. Culturing cells with metformin (50 micromol/l) prevented the HG-induced abnormalities in relaxation without ameliorating depressed peak-shortening amplitudes. Incubation of the cells with metformin also prevented slower intracellular Ca2+ clearing induced by HG. However, the HG-induced relaxation defects were not improved by glyburide (50-300 micromol/l). Interestingly, metformin also improved HG-induced relaxation abnormalities in the absence of insulin, whereas it failed to protect against HG in the presence of the tyrosine kinase inhibitor genistein (50 micromol/l). These data demonstrate that, unlike glyburide, metformin provides cardioprotection against HG-induced abnormalities in myocyte relaxation, perhaps through tyrosine kinase-dependent changes in intracellular Ca2+ handling, independent of its insulin sensitizing action. [ABSTRACT FROM AUTHOR]

The transcriptional nuclear factor (NF)-kappaB can be activated by diverse stimuli such as cytokines, mitogens, oxidative stress, and lipids, leading to the transactivation of several genes that play important roles in the development of atherosclerosis. Because oxidative stress may play a key role in the pathogenesis of diabetic vascular disease, we have examined whether culture of porcine vascular smooth muscle cells (PVSMCs) under high glucose (HG) conditions (25 mmol/l) to simulate the diabetic state can lead to the activation of NF-kappaB, and also whether cytokine- or growth factor-induced NF-kappaB activation is altered by HG culture. We observed that PVSMCs cultured in HG showed significantly greater activation of NF-kappaB in the basal state compared with cells cultured in normal glucose (NG) (5.5 mmol/l). Treatment of the cells with cytokines, such as tumor necrosis factor (TNF)-alpha and interleukin-1beta, or with growth factors, such as platelet-derived growth factor, insulin-like growth factor-I, and epidermal growth factor, all led to NF-kappaB activation in cells cultured in both NG and HG. However, their effects were markedly greater in HG. The augmented TNF-alpha-induced NF-kappaB activation in HG was associated with increased TNF-alpha-mediated transcriptional activation of the vascular cell adhesion molecule-1 promoter. Immunoblotting with an antibody to the p65 subunit of NF-kappaB indicated that the levels of this protein were higher in the nuclear extracts from cells cultured in HG compared with NG. Cells cultured in HG also produced significantly greater amounts of the reactive oxygen species superoxide. HG-induced NF-kappaB activation was inhibited by a protein kinase C inhibitor, calphostin C. These results suggest that hyperglycemia-induced activation of NF-kappaB in VSMCs may be a key mechanism for the accelerated vascular disease observed in diabetes. [ABSTRACT FROM AUTHOR]

Diabetic endothelial dysfunction associated with diminished endothelial nitric oxide (NO) synthase (eNOS) activity accelerates the development of atherosclerosis and cardiomyopathy. However, the approaches to restore eNOS activity and endothelial function in diabetes remain limited. The current study shows that enhanced expression of Krüppel-like factor 2 (KLF2), a shear stress-inducible transcription factor, effectively improves endothelial function through increasing NO bioavailability. KLF2 expression is suppressed in diabetic mouse aortic endothelium. Running exercise and simvastatin treatment induce endothelial KLF2 expression in db/db mice. Adenovirus-mediated endothelium-specific KLF2 overexpression enhances both endothelium-dependent relaxation and flow-mediated dilatation, while it attenuates oxidative stress in diabetic mouse arteries. KLF2 overexpression increases the phosphorylation of eNOS at serine 1177 and eNOS dimerization. RNA-sequencing analysis reveals that KLF2 transcriptionally upregulates genes that are enriched in the cyclic guanosine monophosphate-protein kinase G-signaling pathway, cAMP-signaling pathway, and insulin-signaling pathway, all of which are the upstream regulators of eNOS activity. Activation of the phosphoinositide 3-kinase-Akt pathway and Hsp90 contributes to KLF2-induced increase of eNOS activity. The present results suggest that approaches inducing KLF2 activation, such as physical exercise, are effective to restore eNOS activity against diabetic endothelial dysfunction. [ABSTRACT FROM AUTHOR]

Diabetic retinopathy (DR), one of the most common microangiopathic complications in diabetes, causes severe visual damage among working-age populations. Retinal vascular endothelial cells, the key cell type in DR pathogenesis, are responsible for abnormal retinal angiogenesis in advanced stages of DR. The roles of exosomes in DR have been largely unknown. In this study, we report the first evidence that exosomes derived from the vitreous humor of patients with proliferative DR (PDR-exo) promote proliferation, migration, and tube formation of human retinal vascular endothelial cells (HRVECs). We identified long noncoding RNA (lncRNA) LOC100132249 enrichment in PDR-exo via high-throughput sequencing. This lncRNA, also mainly derived from HRVECs, promoted angiogenesis both in vitro and in vivo. Mechanistically, LOC100132249 acted as a competing endogenous sponge of miRNA-199a-5p (miR-199a-5p), thus regulating the endothelial-mesenchymal transition promoter SNAI1 via activation of the Wnt/β-catenin pathway and ultimately resulting in endothelial dysfunction. In conclusion, our findings underscored the pathogenic role of endothelial-derived exosomes via the LOC100132249/miR-199a-5p/SNAI1 axis in DR angiogenesis and may shed light on new therapeutic strategies for future treatment of DR. [ABSTRACT FROM AUTHOR]

Diabetes is associated with decreased epoxyeicosatrienoic acid (EET) bioavailability and increased levels of glomerular vascular endothelial growth factor A (VEGF-A) expression. We examined whether a soluble epoxide hydrolase inhibitor protects against pathologic changes in diabetic kidney disease and whether the inhibition of the VEGF-A signaling pathway attenuates diabetes-induced glomerular injury. We also aimed to delineate the cross talk between cytochrome P450 2C (CYP2C)–derived EETs and VEGF-A. Streptozotocin-induced type 1 diabetic (T1D) rats were treated with 25 mg/L of 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA) in drinking water for 6 weeks. In parallel experiments, T1D rats were treated with either SU5416 or humanized monoclonal anti–VEGF-A neutralizing antibody for 8 weeks. Following treatment, the rats were euthanized, and kidney cortices were isolated for further analysis. Treatment with AUDA attenuated the diabetes-induced decline in kidney function. Furthermore, treatment with AUDA decreased diabetes-associated oxidative stress and NADPH oxidase activity. Interestingly, the downregulation of CYP2C11-derived EET formation is found to be correlated with the activation of the VEGF-A signaling pathway. In fact, inhibiting VEGF-A using anti-VEGF or SU5416 markedly attenuated diabetes-induced glomerular injury through the inhibition of Nox4-induced reactive oxygen species production. These findings were replicated in vitro in rat and human podocytes cultured in a diabetic milieu. Taken together, our results indicate that hyperglycemia-induced glomerular injury is mediated by the downregulation of CYP2C11-derived EET formation, followed by the activation of VEGF-A signaling and upregulation of Nox4. To our knowledge, this is the first study to highlight VEGF-A as a mechanistic link between CYP2C11-derived EET production and Nox4. Article Highlights: Diabetes is associated with an alteration in cytochrome P450 2C11 (CYP2C11)–derived epoxyeicosatrienoic acid (EET) bioavailability. Decreased CYP2C11-derived EET bioavailability mediates hyperglycemia-induced glomerular injury. Decreased CYP2C11-derived EET bioavailability is associated with increased reactive oxygen species production, NADPH oxidase activity, and Nox4 expression in type 1 diabetes. Decreased CYP2C11-derived EET formation mediates hyperglycemia-induced glomerular injury through the activation of the vascular endothelial growth factor A (VEGF-A) signaling pathway. Inhibiting VEGF signaling using anti-VEGF or SU5416 attenuates type 1 diabetes–induced glomerular injury by decreasing NADPH oxidase activity and NOX4 expression. [ABSTRACT FROM AUTHOR]

Endothelial cell (EC) activation is a crucial determinant of retinal vascular inflammation associated with diabetic retinopathy (DR), a major microvascular complication of diabetes. We previously showed that, similar to abnormal biochemical factors, aberrant mechanical cues in the form of lysyl oxidase (LOX)-dependent subendothelial matrix stiffening also contribute significantly to retinal EC activation in diabetes. Yet, how LOX is itself regulated and precisely how it mechanically controls retinal EC activation in diabetes is poorly understood. Here, we show that high-glucose–induced LOX upregulation in human retinal ECs (HRECs) is mediated by proinflammatory receptor for advanced glycation end products (RAGE). HRECs treated with methylglyoxal (MGO), an active precursor to the advanced glycation end product (AGE) MG-H1, exhibited LOX upregulation that was blocked by a RAGE inhibitor, thus confirming the ability of RAGE to promote LOX expression. Crucially, as a downstream effector of RAGE, LOX was found to mediate both the proinflammatory and matrix remodeling effects of AGE/RAGE, primarily through its ability to crosslink or stiffen matrix. Finally, using decellularized HREC-derived matrices and a mouse model of diabetes, we demonstrate that LOX-dependent matrix stiffening feeds back to enhance RAGE, thereby achieving its autoregulation and proinflammatory effects. Collectively, these findings provide fresh mechanistic insights into the regulation and proinflammatory role of LOX-dependent mechanical cues in diabetes while simultaneously implicating LOX as an alternative (downstream) target to block AGE/RAGE signaling in DR. Article Highlights: We investigated the regulation and proinflammatory role of retinal endothelial lysyl oxidase (LOX) in diabetes. Findings reveal that LOX is upregulated by advanced glycation end products (AGE) and receptor for AGE (RAGE) and mediates AGE/RAGE-induced retinal endothelial cell activation and subendothelial matrix remodeling. We also show that LOX-dependent subendothelial matrix stiffening feeds back to enhance retinal endothelial RAGE. These findings implicate LOX as a key proinflammatory factor and an alternative (downstream) target to block AGE/RAGE signaling in diabetic retinopathy. [ABSTRACT FROM AUTHOR]

Ferroptosis is a newly identified form of regulated cell death that is driven by iron overload and uncontrolled lipid peroxidation, but the role of ferroptosis in cardiac microvascular dysfunction remains unclear. Isorhapontigenin (ISO) is an analog of resveratrol and possesses strong antioxidant capacity and cardiovascular protective effects. Moreover, ISO has been shown to alleviate iron-induced oxidative damage and lipid peroxidation in mitochondria. Therefore, the present study aimed to explore the benefits of ISO treatment on cardiac microvascular dysfunction in diabetes mellitus and the possible mechanisms involved, with a focus on ferroptosis and mitochondria. Our data revealed that ISO treatment improved microvascular density and perfusion in db/db mice by mitigating vascular structural damage, normalizing nitric oxide (NO) production via eNOS activation, and enhancing angiogenetic ability via VEGFR2 phosphorylation. PRDX2 was identified as a downstream target of ISO, and endothelial-specific overexpression of PRDX2 exerted effects on the cardiac microvascular function that were similar to those of ISO treatment. In addition, PRDX2 mediated the inhibitive effects of ISO treatment on ferroptosis by suppressing oxidative stress, iron overload and lipid peroxidation. Further study suggested that mitochondrial dynamics and dysfunction contributed to ferroptosis, and ISO treatment or PRDX2 overexpression attenuated mitochondrial dysfunction via MFN2-dependent mitochondrial dynamics. Moreover, MFN2 overexpression suppressed the mitochondrial translocation of ACSL4, ultimately inhibiting mitochondrial-derived ferroptosis. In contrast, enhancing mitochondrial-derived ferroptosis via ACSL4 abolished the protective effects of ISO treatment on cardiac microcirculation. Taken together, the results of the present work demonstrated the beneficial effects of ISO treatment on cardiac microvascular protection in diabetes mellitus by suppressing mitochondrial-derived ferroptosis through PRDX2-MFN2-ACSL4 pathways. [ABSTRACT FROM AUTHOR]

It is demonstrated that glucose fluctuations, such as postprandial hyperglycemia, and hypoglycemia due to diabetes treatment could be implicated in the development of diabetic complications. There is evidence for the involvement of oxidative stress in the pathogenesis of diabetic neuropathy. It has been elucidated that hyperglycemia induced oxidative stress and caused peripheral nerve dysfunction. However, the effects of hypoglycemia and blood glucose fluctuation on diabetic neuropathy remain unclear. In the present study, we investigated the mechanisms of recurrent short-term hypoglycemia and hyperglycemia on apoptosis and oxidative stress in Schwann cells, and we also examined the involvement of polyol pathway and endoplasmic reticulum (ER) stress pathway in these abnormalities. Immortalized adult mouse Schwann cells were exposed to five different glucose treatments over 3 days: 1) normal glucose (NG), 2) constant low glucose (LG), 3) constant high glucose (HG), 4) intermittent low glucose (ILG; 1 h three times per day), 5) intermittent high glucose (IHG; 1 h three times per day). Cell viability was reduced not only HG, but also LG. Furthermore, either IHG or ILG also decreased cell viabilities. TBARS levels were increased by HG, LG, IHG, and ILG. High glucose (HG and IHG) and low glucose (LG and ILG) increased the expression of cleaved caspase-3 and reduced that of Bcl-2. Epalrestat, an aldose reductase inhibitor, as well as 4-PBA, an ER stress inhibitor, recovered cell death and oxidative stress which were induced by LG, HG, ILG and IHG. These results suggest that intermittent hypoglycemia and hyperglycemia enhanced apoptosis and oxidative stress through both polyol pathway and ER stress pathway in Schwann cells, and that not only hyperglycemia, but also hypoglycemia and glucose fluctuations might cause nerve dysfunction in diabetes, resulting in the onset and progression of diabetic neuropathy. Disclosure: A. Kato: None. Y. Tatsumi: None. H. Yako: None. T. Himeno: None. M. Kondo: None. Y. Kato: Speaker's Bureau; Self; Merck & Co., Inc. H. Kamiya: Speaker's Bureau; Self; Astellas Pharma Inc., Eli Lilly Japan K.K., MSD K.K., Novartis Pharma K.K., Novo Nordisk Oharma Ltd., Ono Pharmaceutical Co., Ltd., Sanofi K.K. K. Sango: None. J. Nakamura: Research Support; Self; Astellas Pharma Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Daiichi Sankyo Company, Limited, Eli Lilly and Company, Japan Tobacco Inc., Kissei Pharmaceutical Co., Ltd., Merck Sharp & Dohme Corp., Novartis Pharmaceuticals Corporation, Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sanofi K.K., Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited. Speaker's Bureau; Self; Astellas Pharma Inc., AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Daiichi Sankyo Company, Limited, Eli Lilly and Company, Kowa Pharmaceu. Co. Ltd., Merck Sharp & Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Novartis Pharmaceuticals Corporation, Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sanofi K.K., Takeda Pharmaceutical Company Limited, Terumo Medical Corporation. K. Kato: None. Funding: Japan Society for the Promotion of Science [ABSTRACT FROM AUTHOR]

Hyperglycemia-induced inflammation and apoptosis have important roles in the pathogenesis of diabetic cardiomyopathy. We recently found that a novel curcumin derivative, C66, is able to reduce the high glucose (HG)-induced inflammatory response. This study was designed to investigate the protective effects on diabetic cardiomyopathy and its underlying mechanisms. Pretreatment with C66 significantly reduced HG-induced overexpression of inflammatory cytokines via inactivation of nuclear factor-[kappa]B in both H9c2 cells and neonatal cardiomyocytes. Furthermore, we showed that the inhibition of Jun NH2-terminal kinase (JNK) phosphorylation contributed to the protection of C66 from inflammation and cell apoptosis, which was validated by the use of SP600125 and dominant-negative JNK. The molecular docking and kinase activity assay confirmed direct binding of C66 to and inhibition of JNK. In mice with type 1 diabetes, the administration of C66 or SP600125 at 5 mg/kg significantly decreased the levels of plasma and cardiac tumor necrosis factor-[alpha], accompanied by decreasing cardiac apoptosis, and, finally, improved histological abnormalities, fibrosis, and cardiac dysfunction without affecting hyperglycemia. Thus, this work demonstrated the therapeutic potential of the JNK-targeting compound C66 for the treatment of diabetic cardiomyopathy. Importantly, we indicated a critical role of JNK in diabetic heart injury, and suggested that JNK inhibition may be a feasible strategy for treating diabetic cardiomyopathy. [ABSTRACT FROM AUTHOR]

Impaired pancreatic β-cell function and insulin secretion are hallmarks of type 2 diabetes. miRNAs are short, noncoding RNAs that silence gene expression vital for the development and function of β cells. We have previously shown that β cell-specific deletion of the important energy sensor AMP-activated protein kinase (AMPK) results in increased miR-125b-5p levels. Nevertheless, the function of this miRNA in β cells is unclear. We hypothesized that miR-125b-5p expression is regulated by glucose and that this miRNA mediates some of the deleterious effects of hyperglycemia in β cells. Here, we show that islet miR-125b-5p expression is upregulated by glucose in an AMPK-dependent manner and that short-term miR-125b-5p overexpression impairs glucose-stimulated insulin secretion (GSIS) in the mouse insulinoma MIN6 cells and in human islets. An unbiased, high-throughput screen in MIN6 cells identified multiple miR-125b-5p targets, including the transporter of lysosomal hydrolases M6pr and the mitochondrial fission regulator Mtfp1. Inactivation of miR-125b-5p in the human β-cell line EndoCβ-H1 shortened mitochondria and enhanced GSIS, whereas mice overexpressing miR-125b-5p selectively in β cells (MIR125B-Tg) were hyperglycemic and glucose intolerant. MIR125B-Tg β cells contained enlarged lysosomal structures and had reduced insulin content and secretion. Collectively, we identify miR-125b as a glucose-controlled regulator of organelle dynamics that modulates insulin secretion. [ABSTRACT FROM AUTHOR]

Excessive production of renal reactive oxygen species (ROS) plays a major role in diabetic kidney disease (DKD). Here, we provide key findings demonstrating the predominant pathological role of the pro-oxidant enzyme NADPH oxidase 5 (NOX5) in DKD, independent of the previously characterized NOX4 pathway. In patients with diabetes, we found increased expression of renal NOX5 in association with enhanced ROS formation and upregulation of ROS-sensitive factors early growth response 1 (EGR-1), protein kinase C-α (PKC-α), and a key metabolic gene involved in redox balance, thioredoxin-interacting protein (TXNIP). In preclinical models of DKD, overexpression of NOX5 in Nox4-deficient mice enhances kidney damage by increasing albuminuria and augmenting renal fibrosis and inflammation via enhanced ROS formation and the modulation of EGR1, TXNIP, ERK1/2, PKC-α, and PKC-ε. In addition, the only first-in-class NOX inhibitor, GKT137831, appears to be ineffective in the presence of NOX5 expression in diabetes. In vitro, silencing of NOX5 in human mesangial cells attenuated upregulation of EGR1, PKC-α, and TXNIP induced by high glucose levels, as well as markers of inflammation (TLR4 and MCP-1) and fibrosis (CTGF and collagens I and III) via reduction in ROS formation. Collectively, these findings identify NOX5 as a superior target in human DKD compared with other NOX isoforms such as NOX4, which may have been overinterpreted in previous rodent studies. [ABSTRACT FROM AUTHOR]

Mitochondria-associated endoplasmic reticulum membrane (MAM) may have a role in tubular injury in diabetic nephropathy (DN), but the precise mechanism remains unclear. Here, we demonstrate that the expression of phosphofurin acidic cluster sorting protein 2 (PACS-2), a critical regulator of MAM formation, is significantly decreased in renal tubules of patients with DN, and PACS-2 expression is positively correlated with renal function and negatively correlated with degrees of tubulointerstitial lesions. Conditional deletion of Pacs-2 in proximal tubules (PTs) aggravates albuminuria and tubular injury in a streptozotocin-induced mouse model of diabetes. Mitochondrial fragmentation, MAM disruption, and defective mitophagy accompanied by altered expression of mitochondrial dynamics and mitophagic proteins, including Drp1 and Becn1, are observed in tubules of diabetic mice; these changes are more pronounced in PT-specific Pacs-2 knockout mice. In vitro, overexpression of PACS-2 in HK-2 cells alleviates excessive mitochondrial fission induced by high glucose concentrations through blocking mitochondrial recruitment of DRP1 and subsequently restores MAM integrity and enhances mitophagy. Mechanistically, PACS-2 binds to BECN1 and mediates the relocalization of BECN1 to MAM, where it promotes the formation of mitophagosome. Together, these data highlight an important but previously unrecognized role of PACS-2 in ameliorating tubular injury in DN by facilitating MAM formation and mitophagy. [ABSTRACT FROM AUTHOR]

Diabetes mellitus (DM) affects the biology of multipotent cardiac stem/progenitor cells (CSCs) and adult myocardial regeneration. We assessed the hypothesis that senescence and senescence-associated secretory phenotype (SASP) are main mechanisms of cardiac degenerative defect in DM. Accordingly, we tested whether ablation of senescent CSCs would rescue the cardiac regenerative/reparative defect imposed by DM. We obtained cardiac tissue from nonaged (50- to 64-year-old) patients with type 2 diabetes mellitus (T2DM) and without DM (NDM) and postinfarct cardiomyopathy undergoing cardiac surgery. A higher reactive oxygen species production in T2DM was associated with an increased number of senescent/dysfunctional T2DM-human CSCs (hCSCs) with reduced proliferation, clonogenesis/spherogenesis, and myogenic differentiation versus NDM-hCSCs in vitro. T2DM-hCSCs showed a defined pathologic SASP. A combination of two senolytics, dasatinib (D) and quercetin (Q), cleared senescent T2DM-hCSCs in vitro, restoring their expansion and myogenic differentiation capacities. In a T2DM model in young mice, diabetic status per se (independently of ischemia and age) caused CSC senescence coupled with myocardial pathologic remodeling and cardiac dysfunction. D + Q treatment efficiently eliminated senescent cells, rescuing CSC function, which resulted in functional myocardial repair/regeneration, improving cardiac function in murine DM. In conclusion, DM hampers CSC biology, inhibiting CSCs' regenerative potential through the induction of cellular senescence and SASP independently from aging. Senolytics clear senescence, abrogating the SASP and restoring a fully proliferative/differentiation-competent hCSC pool in T2DM with normalization of cardiac function. [ABSTRACT FROM AUTHOR]

Protein kinase C (PKC)β2 is preferably overexpressed in the diabetic myocardium, which induces cardiomyocyte hypertrophy and contributes to diabetic cardiomyopathy, but the underlying mechanisms are incompletely understood. Caveolae are critical in signal transduction of PKC isoforms in cardiomyocytes. Caveolin (Cav)-3, the cardiomyocyte-specific caveolar structural protein isoform, is decreased in the diabetic heart. The current study determined whether PKCβ2 activation affects caveolae and Cav-3 expression. Immunoprecipitation and immunofluorescence analysis revealed that high glucose (HG) increased the association and colocalization of PKCβ2 and Cav-3 in isolated cardiomyocytes. Disruption of caveolae by methyl-β-cyclodextrin or Cav-3 small interfering (si)RNA transfection prevented HG-induced PKCβ2 phosphorylation. Inhibition of PKCβ2 activation by compound CGP53353 or knockdown of PKCβ2 expression via siRNA attenuated the reductions of Cav-3 expression and Akt/endothelial nitric oxide synthase (eNOS) phosphorylation in cardiomyocytes exposed to HG. LY333531 treatment (for a duration of 4 weeks) prevented excessive PKCβ2 activation and attenuated cardiac diastolic dysfunction in rats with streptozotocin-induced diabetes. LY333531 suppressed the decreased expression of myocardial NO, Cav-3, phosphorylated (p)-Akt, and p-eNOS and also mitigated the augmentation of O2(-), nitrotyrosine, Cav-1, and iNOS expression. In conclusion, hyperglycemia-induced PKCβ2 activation requires caveolae and is associated with reduced Cav-3 expression in the diabetic heart. Prevention of excessive PKCβ2 activation attenuated cardiac diastolic dysfunction by restoring Cav-3 expression and subsequently rescuing Akt/eNOS/NO signaling. [ABSTRACT FROM AUTHOR]