Background/Aims: Diabetic non-healing skin ulcers represent a serious challenge in clinical practice, in which the hyperglycemia-induced disturbance of angiogenesis, and endothelial dysfunction play a crucial role. Resveratrol (RES), a silent information regulator 1 (SIRT1) agonist, can improve endothelial function and has strong proangiogenic properties, and has thus become a research focus for the treatment of diabetic non-healing skin ulcers; however, the underlying mechanism by which RES regulates these processes remains unclear. Therefore, the present study was intended to determine if RES exerts its observed protective role in diabetic wound healing by alleviating hyperglycemia-induced endothelial dysfunction and the disturbance of angiogenesis. Methods:We investigated the effects of RES on cell migration, cell proliferation, apoptosis, tube formation, and the underlying molecular mechanisms in 33 mM high glucose-stimulated human umbilical vein endothelial cells (HUVECs) by semiquantitative RT-PCR, western blot analysis, terminal deoxynucleotidyl transferasemediated dUTP nick end labeling (TUNEL) staining, and immunofluorescence in vitro. We further explored the role of RES on endothelial dysfunction and wound healing disturbance in db/db mice by TUNEL staining, immunofluorescence, and photography in vivo. Results:We observed an obvious inhibition of hyperglycemia-triggered endothelial dysfunction and a disturbance of angiogenesis, followed by the promotion of diabetic wound healing via RES, along with restoration of the activity of the hyperglycemiaimpaired SIRT1 signaling pathway. Pretreatment with EX-527, a SIRT1 inhibitor, abolished the RES-mediated endothelial protection and pro-angiogenesis action, and then delayed diabetic wound healing. Furthermore, examination of the overexpression of forkhead box O1 (FOXO1), a transcription factor substrate of SIRT1, in HUVECs Frontiers in Pharmacology | www.frontiersin.org April 2019 | Volume 10 | Article 421 Huang et al.Resveratrol Promotes Diabetic Wound Healing and db/db mice revealed that RES activated SIRT1 to restore hyperglycemia-triggered endothelial dysfunction and disturbance of angiogenesis, followed by the promotion of diabetic wound healing in a c-Myc-dependent manner. Pretreatment with 10058-F4, a c-Myc inhibitor, repressed RES-mediated endothelial protection, angiogenesis, and diabetic wound healing. Conclusion:Our findings indicate that the positive role of RES in diabetic wound healing via its SIRT1-dependent endothelial protection and pro-angiogenic effects involves the inhibition of FOXO1 and the de-repression of c-Myc expression.

BackgroundLipotoxicity is a key feature of the pathogenesis of diabetic kidney disease, and is attributed to excessive lipid accumulation (hyperlipidemia). Increasing evidence suggests that fibroblast growth factor (FGF)21 has a crucial role in lipid metabolism under diabetic conditions.ObjectiveThe present study investigated whether FGF21 can prevent hyperlipidemia- or diabetes-induced renal damage, and if so, the possible mechanism.MethodsMice were injected with free fatty acids (FFAs, 10 mg/10 g body weight) or streptozotocin (150 mg/kg) to establish a lipotoxic model or type 1 diabetic model, respectively. Simultaneously the mice were treated with FGF21 (100 µg/kg) for 10 or 80 days. The kidney weight-to-tibia length ratio and renal function were assessed. Systematic and renal lipid levels were detected by ELISA and Oil Red O staining. Renal apoptosis was examined by TUNEL assay. Inflammation, oxidative stress, and fibrosis were assessed by Western blot.ResultsAcute FFA administration and chronic diabetes were associated with lower kidney-to-tibia length ratio, higher lipid levels, severe renal apoptosis and renal dysfunction. Obvious inflammation, oxidative stress and fibrosis also observed in the kidney of both mice models. Deletion of the fgf21 gene further enhanced the above pathological changes, which were significantly prevented by administration of exogenous FGF21.ConclusionThese results suggest that FFA administration and diabetes induced renal damage, which was further enhanced in FGF21 knock-out mice. Administration of FGF21 significantly prevented both FFA- and diabetes-induced renal damage partially by decreasing renal lipid accumulation and suppressing inflammation, oxidative stress, and fibrosis.

Studies regarding macroautophagic/autophagic regulation in endothelial cells (ECs) under diabetic conditions are very limited. Clinical evidence establishes an endothelial protective effect of metformin, but the underlying mechanisms remain unclear. We aimed to investigate whether metformin exerts its protective role against hyperglycemia-induced endothelial impairment through the autophagy machinery. db/db mice were treated with intravitreal metformin injections. Human umbilical vein endothelial cells (HUVECs) were cultured either in normal glucose (NG, 5.5 mM) or high glucose (HG, 33 mM) medium in the presence or absence of metformin for 72 h. We observed an obvious inhibition of hyperglycemia-triggered autophagosome synthesis in both the diabetic retinal vasculature and cultured HUVECs by metformin, along with restoration of hyperglycemia-impaired Hedgehog (Hh) pathway activity. Specifically, deletion of ATG7 in retinal vascular ECs of db/db mice and cultured HUVECs indicated a detrimental role of autophagy in hyperglycemia-induced endothelial dysfunction. Pretreatment with GANT61, a Hh pathway inhibitor, abolished the metformin-mediated downregulation of autophagy and endothelial protective action. Furthermore, GLI-family (transcription factors of the Hh pathway) knockdown in HUVECs and retinal vasculature revealed that downregulation of hyperglycemia-activated autophagy by the metformin-mediated Hh pathway activation was GLI1 dependent. Mechanistically, GLI1 knockdown-triggered autophagy was related to upregulation of BNIP3, which subsequently disrupted the association of BECN1/Beclin 1 and BCL2. The role of BNIP3 in BECN1 dissociation from BCL2 was further confirmed by BNIP3 overexpression or BNIP3 RNAi. Taken together, the endothelial protective effect of metformin under hyperglycemia conditions could be partly attributed to its role in downregulating autophagy via Hh pathway activation. Abbreviations: 3-MA = 3-methyladenine; 8×GLI BS-FL = 8×GLI-binding site firefly luciferase; AAV = adenoassociated virus; AAV-Cdh5-sh-Atg7 = AAV vectors carrying shRNA against murine Atg7 under control of murine Cdh5 promoter; AAV-Cdh5-sh-Gli1 = AAV vectors carrying shRNA against murine Gli1 under control of murine Cdh5 promoter; AAV-Cdh5-Gli1 = AAV vectors carrying murine Gli1 cDNA under the control of murine Cdh5 core promoter; ACAC = acetyl-CoA carboxylase; Ad-BNIP3 = adenoviruses harboring human BNIP3`; Ad-GLI1 = adenoviruses harboring human GLI1; Ad-sh-ATG7 = adenoviruses harboring shRNA against human ATG7; Ad-sh-BNIP3 = adenoviruses harboring shRNA against human BNIP3; Ad-sh-GLI = adenoviruses harboring shRNA against human GLI; AGEs = advanced glycation end products; ATG = autophagy-related; atg7 flox/flox mice = mice bearing an Atg7 flox allele, in which exon 14 of the Atg7 gene is flanked by 2 loxP sites; BafA1 = bafilomycin A 1 ; BECN1 = beclin 1; CDH5/VE-cadherin = cadherin 5; CASP3 = caspase 3; CASP8 = caspase 8; CASP9 = caspase 9; ECs = endothelial cells; GAPDH = glyceraldehyde-3-phosphate dehydrogenase; GCL = g...

BackgroundDyslipidemia and lipotoxicity-induced insulin resistance, inflammation and oxidative stress are the key pathogeneses of renal damage in type 2 diabetes. Increasing evidence shows that whole-body low dose radiation (LDR) plays a critical role in attenuating insulin resistance, inflammation and oxidative stress.ObjectiveThe aims of the present study were to investigate whether LDR can prevent type 2 diabetes-induced renal damage and the underlying mechanisms.MethodsMice were fed with a high-fat diet (HFD, 40% of calories from fat) for 12 weeks to induce obesity followed by a single intraperitoneal injection of streptozotocin (STZ, 50 mg/kg) to develop a type 2 diabetic mouse model. The mice were exposed to LDR at different doses (25, 50 and 75 mGy) for 4 or 8 weeks along with HFD treatment. At each time-point, the kidney weight, renal function, blood glucose level and insulin resistance were examined. The pathological changes, renal lipid profiles, inflammation, oxidative stress and fibrosis were also measured.ResultsHFD/STZ-induced type 2 diabetic mice exhibited severe pathological changes in the kidney and renal dysfunction. Exposure of the mice to LDR for 4 weeks, especially at 50 and 75 mGy, significantly improved lipid profiles, insulin sensitivity and protein kinase B activation, meanwhile, attenuated inflammation and oxidative stress in the diabetic kidney. The LDR-induced anti-oxidative effect was associated with up-regulation of renal nuclear factor E2-related factor-2 (Nrf-2) expression and function. However, the above beneficial effects were weakened once LDR treatment was extended to 8 weeks.ConclusionThese results suggest that LDR exposure significantly prevented type 2 diabetes-induced kidney injury characterized by renal dysfunction and pathological changes. The protective mechanisms of LDR are complicated but may be mainly attributed to the attenuation of dyslipidemia and the subsequent lipotoxicity-induced insulin resistance, inflammation and oxidative stress.

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␦-Catenin was first identified because of its interaction with presenilin-1, and its aberrant expression has been reported in various human tumors and in patients with Cri-du-Chat syndrome, a form of mental retardation. However, the mechanism whereby ␦-catenin is regulated in cells has not been fully elucidated. We investigated the possibility that glycogen-synthase kinase-3 (GSK-3) phosphorylates ␦-catenin and thus affects its stability. Initially, we found that the level of ␦-catenin was greater and the half-life of ␦-catenin was longer in GSK-3␤ fibroblasts than those in GSK-3␤؉/؉ fibroblasts. Furthermore, four different approaches designed to specifically inhibit GSK-3 activity, i.e. GSK-3-specific chemical inhibitors, Wnt-3a conditioned media, small interfering RNAs, and GSK-3␣ and -3␤ kinase dead constructs, consistently showed that the levels of endogenous ␦-catenin in CWR22Rv-1 prostate carcinoma cells and primary cortical neurons were increased by inhibiting GSK-3 activity. In addition, it was found that both GSK-3␣ and -3␤ interact with and phosphorylate ␦-catenin. The phosphorylation of ⌬C207-␦-catenin (lacking 207 C-terminal residues) and T1078A ␦-catenin by GSK-3 was noticeably reduced compared with that of wild type ␦-catenin, and the data from liquid chromatography-tandem mass spectrometry analyses suggest that the Thr 1078 residue of ␦-catenin is one of the GSK-3 phosphorylation sites. Treatment with MG132 or ALLN, specific inhibitors of proteosome-dependent proteolysis, increased ␦-catenin levels and caused an accumulation of ubiquitinated ␦-catenin. It was also found that GSK-3 triggers the ubiquitination of ␦-catenin. These results suggest that GSK-3 interacts with and phosphorylates ␦-catenin and thereby negatively affects its stability by enabling its ubiquitination/proteosome-mediated proteolysis.

Skin wound healing is a complex process requiring the coordinated behavior of many cell types, especially in the proliferation and migration of fibroblasts. Basic fibroblast growth factor (bFGF) is a member of the FGF family that promotes fibroblast migration, but the underlying molecular mechanism remains elusive. The present RNA sequencing study showed that the expression levels of several canonical Wnt pathway genes, including Wnt2b, Wnt3, Wnt11, T-cell factor 7 (TCF7), and Frizzled 8 (FZD8) were modified by bFGF stimulation in fibroblasts. Enzyme-linked immunosorbent assay (ELISA) analysis also showed that Wnt pathway was activated under bFGF treatment. Furthermore, treatment of fibroblasts with lithium chloride or IWR-1, an inducer and inhibitor of the Wnt signaling pathway, respectively, promoted and inhibited cell migration. Also, levels of cytosolic glycogen synthase kinase 3 beta phosphorylated at serine9 (pGSK3β Ser9) and nuclear β-catenin were increased upon exposure to bFGF. Molecular and biochemical assays indicated that phosphoinositide 3-kinase (PI3K) signaling activated the GSK3β/β-catenin/Wnt signaling pathway via activation of c-Jun N-terminal kinase (JNK), suggesting that PI3K and JNK act at the upstream of β-catenin. In contrast, knock-down of β-catenin delayed fibroblast cell migration even under bFGF stimulation. RNA sequencing analysis of β-catenin knock-down fibroblasts demonstrated that β-catenin positively regulated the transcription of bFGF and FGF21. Moreover, FGF21 treatment activated AKT and JNK, and accelerated fibroblast migration to a similar extent as bFGF does. In addition, ELISA analysis demonstrated that both of bFGF and FGF21 were auto secretion factor and be regulated by Wnt pathway stimulators. Taken together, our analyses define a feedback regulatory loop between bFGF (FGF21) and Wnt signaling acting through β-catenin in skin fibroblasts.

Fibroblast growth factor (FGF)-21 is a novel regulator of insulin-independent glucose transport in 3T3-L1 adipocytes and has glucose and triglyceride lowering effects in rodent models of diabetes. In this study, we found that FGF-21 can significantly attenuate ischemia-reperfusion (I/R) induced damage in H9c2 cells (rat heart). However, it is unclear which signal transduction pathway is involved in the cardioprotective effect of FGF-21. Thus, this study was designed to investigate the potential mechanism induced by FGF-21. The results showed that FGF-21 treatment prevented the oxidative stress and apoptosis associated with I/R damage by reducing the levels of superoxide anions, inhibiting glycogen synthase kinase (GSK) 3β by activating Akt phosphorylation, and recovering the levels of ATP synthase pyruvate kinase isozymes M1 and protein kinase C, thereby improving energy supply. In summary, we conclude that FGF-21 protects H9c2 cells against I/R injury mainly through the Akt-GSK-3β-caspase-3 dependent pathway, preventing oxidative stress, and recovery of the energy supply.