Weitao Cong

Attenuation of hyperlipidemia- and diabetes-induced early-stage apoptosis and late-stage renal dysfunction via administration of fibroblast growth factor-21 is associated with suppression of renal inflammation.

Background Lipotoxicity 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. Objective The present study investigated whether FGF21 can prevent hyperlipidemia- or diabetes-induced renal damage, and if so, the possible mechanism. Methods Mice 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. Results Acute 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. Conclusion These 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.

GSK-3 Phosphorylates δ-Catenin and Negatively Regulates Its Stability via Ubiquitination/Proteosome-mediated Proteolysis

δ-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 δ-catenin is regulated in cells has not been fully elucidated. We investigated the possibility that glycogen-synthase kinase-3 (GSK-3) phosphorylates δ-catenin and thus affects its stability. Initially, we found that the level of δ-catenin was greater and the half-life of δ-catenin was longer in GSK-3β−/− fibroblasts than those in GSK-3β+/+ 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-3α and -3β kinase dead constructs, consistently showed that the levels of endogenous δ-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-3α and -3β interact with and phosphorylate δ-catenin. The phosphorylation of ΔC207-δ-catenin (lacking 207 C-terminal residues) and T1078A δ-catenin by GSK-3 was noticeably reduced compared with that of wild type δ-catenin, and the data from liquid chromatography-tandem mass spectrometry analyses suggest that the Thr1078 residue of δ-catenin is one of the GSK-3 phosphorylation sites. Treatment with MG132 or ALLN, specific inhibitors of proteosome-dependent proteolysis, increased δ-catenin levels and caused an accumulation of ubiquitinated δ-catenin. It was also found that GSK-3 triggers the ubiquitination of δ-catenin. These results suggest that GSK-3 interacts with and phosphorylates δ-catenin and thereby negatively affects its stability by enabling its ubiquitination/proteosome-mediated proteolysis.

Multiple low-dose radiation prevents type 2 diabetes-induced renal damage through attenuation of dyslipidemia and insulin resistance and subsequent renal inflammation and oxidative stress.

Background Dyslipidemia 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. Objective The aims of the present study were to investigate whether LDR can prevent type 2 diabetes-induced renal damage and the underlying mechanisms. Methods Mice 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. Results HFD/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. Conclusion These 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.

Metallothionein prevents cardiac pathological changes in diabetes by modulating nitration and inactivation of cardiac ATP synthase.

Mitochondrial ATP production is the main energy source for the cell. Diabetes reduces the efficient generation of ATP, possibly due to the inactivation of ATP synthase. However, the exact mechanism by which diabetes induces inactivation of ATP synthase remains unknown, as well as whether such inactivation has a role in the development of pathological abnormalities of the diabetic heart. To address these issues, we used cardiac metallothionein-transgenic (MT-TG) and wild-type (WT) mice with streptozotocin-induced diabetes, since we have demonstrated previously that diabetes-induced cardiac damage and remodeling were found in WT diabetic mice, but not in MT-TG diabetic mice. Immunohistochemical and biochemical assays were used to compare pathological and biochemical changes of the heart between MT-TG and WT diabetic mice, and a proteomic assay to evaluate ATP synthase expression and tyrosine nitration, with its activity. LC/MS analysis revealed that diabetes increased tyrosine nitration of the ATP synthase α subunit at Tyr(271), Tyr(311), and Tyr(476), and the β subunit at Tyr(269) and Tyr(508), and also significantly reduced ATP synthase activity by ~32%. These changes were not observed in MT-TG diabetic mice. Furthermore, parallel experiments with induced expression of cardiac MT by zinc supplementation in diabetic mice produced similar effects. These results suggest that MT can preserve ATP synthase activity in streptozotocin-induced diabetes, probably through the inhibition of ATP synthase nitration.

An improved silver stain for the visualization of lipopolysaccharides on polyacrylamide gels.

A sensitive, brief, and user‐friendly silver stain to meet the needs in high‐efficiency detection of lipopolysaccharides (LPS) on polyacrylamide gels is described. In this study, the most commonly used formaldehyde‐based LPS silver stain, which is potentially hazardous to the operator, is replaced by ascorbic acid (Vc) in alkaline sodium thiosulfate solution. It takes only about 35 min to complete all the protocol, with a detection limit of 4 ng of total LPS. The results indicate that this user‐friendly method could be a good choice for LPS visualization on polyacrylamide gels.

The activation of the NF-κB-JNK pathway is independent of the PI3K-Rac1-JNK pathway involved in the bFGF-regulated human fibroblast cell migration

BACKGROUND Skin wound healing is a complex process that repairs multiple organ-tissues. Fibroblasts are key players of skin cells, whose migration is important during wound healing process. bFGF has shown a great efficacy to promote cell migration, but the precise mechanism by which bFGF regulates cell migration remains elusive. OBJECTIVE The aim of this study was to find bFGF-regulated gene pools and further identify target molecules that participated in human fibroblast cell migration. METHODS Skin primary fibroblasts and rat skin wound model were used to demonstrate the novel mechanism of bFGF regulating cell migration to accelerate wound healing. Cell migration was determined using the wound healing scratch assay. The differentially expressed genes and numerous biochemical pathways after bFGF treatment were identified by RNA-Seq analysis, and differentially expressed genes were further verified by qRT-PCR. siRNA duplex target to interfering the expression of PI3-kinase (p110α) was transformed into NIH/3T3 cells. Western blotting analysis was used to determine marker protein expressions. The invasive activity of fibroblasts was measured using 3D spheroid cell invasion assay. RESULTS RNA-Seq analysis identified numerous biochemical pathways including the NF-κB pathway under the control of FGF signaling. bFGF negatively regulates the phosphorylation of IκB-α, the most well studied NF-κB signaling regulator while bFGF induces JNK phosphorylation. Application of Bay11-7082, a representative NF-κB inhibitor promoted cell migration, invasion and enhanced the JNKs phosphorylation. However, inhibition of JNKs blocked cell migration when NF-κB is inhibited. Moreover, application of the PI3K inhibitor LY294002 together with Bay11-7082 maintained normal cell migration and knocking-down PI3K (p110α) by a specific siRNA inhibited JNKs phosphorylation while maintaining normal IκBα phosphorylation, indicating that PI3K and NF-κB signaling independently regulate JNKs activation. In addition, administration of bFGF or Bay11-7082 promoted rat skin wound repair and accelerated the invasion of fibroblasts. CONCLUSION This study sheds light on the mode of action of bFGF and identifies that the NF-κB-JNKs pathway is independent of the PI3K-JNKs pathway to accelerate fibroblast migration. In addition, bFGF and the relief of inflammation could be a favorable therapeutic approach for skin wound healing.

A novel targeted proteomics method for identification and relative quantitation of difference in nitration degree of OGDH between healthy and diabetic mouse

For analysis of nitration modification of α oxoglutarate dehydrogenase (α‐OGDH) induced by diabetes, a targeted proteomics strategy was developed through the use of Skyline. All peptides containing Y and W of the target proteins were nitrated in silico and output to produce parallel reaction monitoring (PRM) or SRM method for nitration analysis. A nitrated casein mixture was used as standard protein to assess the feasibility of this method. The results demonstrated the availability of this strategy for nitration identification, and subsequently this method was used to analyze the nitration of α‐OGDH from myocardial tissue extracts of diabetic mouse. The PRM method was primarily generated by Skyline for identification of the actual nitrated peptides from all possible nitrated peptides of α‐OGDH due to the complexity of α‐OGDH. The PRM‐based data were analyzed by SEQUEST, and transitions of the identified nitrated peptides were used to develop an SRM method for relative quantitation of nitration degree. The nitration degree of α‐OGDH for diabetic mouse is higher than that for control mouse, indicating that α‐OGDH of the diabetic mouse suffered from more intense oxidative damage. We believe that this approach for obtaining information regarding nitration will facilitate the study of other PTMs in complex mixtures.

Highly Sensitive Method for Specific, Brief, and Economical Detection of Glycoproteins in Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis by the Synthesis of a New Hydrazide Derivative

A new hydrazide derivative was synthesized and used for the first time as a specific, brief, and economical probe to selectively visualize glycoproteins in 1-D and 2-D sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with high sensitivity. The detection limit of the newly developed staining method is 2- and 4-fold higher than that of the widely used Pro-Q Emerald 300 and 488 stains, respectively.

Metallothionein prevents diabetes-induced cardiac pathological changes, likely via the inhibition of succinyl-CoA:3-ketoacid coenzyme A transferase-1 nitration at Trp374

We previously demonstrated that metallothionein (MT)-mediated protection from diabetes-induced pathological changes in cardiac tissues is related to suppression of superoxide generation and protein nitration. The present study investigated which diabetes-nitrated protein(s) mediate the development of these pathological changes by identifying the panel of nitrated proteins present in diabetic hearts of wild-type (WT) mice and not in those of cardiac-specific MT-overexpressing transgenic (MT-TG) mice. At 2, 4, 8, and 16 wk after streptozotocin induction of diabetes, histopathological examination of the WT and MT-TG diabetic hearts revealed cardiac structure derangement and remodeling, significantly increased superoxide generation, and 3-nitrotyrosine accumulation. A nitrated protein of 58 kDa, succinyl-CoA:3-ketoacid CoA transferase-1 (SCOT), was identified by mass spectrometry. Although total SCOT expression was not significantly different between the two types of mice, the diabetic WT hearts showed significantly increased nitration content and dramatically decreased catalyzing activity of SCOT. Although SCOT nitration sites were identified at Tyr(76), Tyr(117), Tyr(135), Tyr(226), Tyr(368), and Trp(374), only Tyr(76) and Trp(374) were found to be located in the active site by three-dimensional structure modeling. However, only Trp(374) showed a significantly different nitration level between the WT and MT-TG diabetic hearts. These results suggest that MT prevention of diabetes-induced pathological changes in cardiac tissues is most likely mediated by suppression of SCOT nitration at Trp(374).

High-throughput negative detection of SDS-PAGE separated proteins and its application for proteomics

A negative detection method for proteins on SDS‐PAGE is described. In this method, Eosin Y (EY) was selectively precipitated in the gel background, which is absent from those zones where proteins are located through the formation of a stable water‐soluble protein–dye complex. Negative staining of proteins using EY, allows high‐sensitivity, low‐cost, and simple protocol. The new described method takes less than an hour to complete all the protocol, with a detection limit of 0.5 ng of single protein band. Comparing with imidazole‐zinc negative stain, EY dye provides broader linear dynamic range, higher sensitivity and reproducibility, and better obvious contrast between the protein bands or spots and background. Furthermore, the novel technique developed here presented a real practical method for simultaneous processing of multiple gels, which makes it possible to perform high‐throughput staining for proteome research. Additionally, we have also compared the influence of staining method on the quality of mass spectra by PMF.