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.
L. Metallothionein prevents diabetes-induced cardiac pathological changes, likely via the inhibition of succinyl-CoA:3-ketoacid coenzyme A transferase-1 nitration at Trp 374 . Am J Physiol Endocrinol Metab 304: E826 -E835, 2013. First published February 26, 2013 doi:10.1152/ajpendo.00570.2012.-We previously demonstrated that metallothionein (MT)-mediated protection from diabetesinduced 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 diabetes; cardiac pathological changes; metallothionein; SCOT; 3-nitrotyrosine DIABETES HAS BECOME one of the most prolific public health issues worldwide (25). Mechanistic studies have revealed that oxidative and nitrosative stress are major causes of diabetes and its complications (4, 13). Overproduced superoxide has been shown in diabetic individuals to interact with nitric oxide, forming the peroxynitrite (ONOO Ϫ ) free radical that mediates biomacromolecule nitration and can ultimately lead to organ dysfunction. The covalent product of tyrosine nitration, 3-nitrotyrosine (3-NT), serves as an index of peroxynitrite-induced protein damage and is used in clinic as a convenient quantitative biomarker of cardiovascular risk (measured in free and protein-bound forms in human plasma) (23). Moreover, overproduction of superoxide and associated peroxynitrite has been implicated in the development of diabetic cardiomyopathy (4, 20, 28). Therefore, a promising approach to prevent the development of diabetic complications will include the prevention of oxidative and nitrosative stress (2).The well-established and widely used animal model of type 1 diabetes induced by streptozotocin (STZ) has several nitrated molecules in the cardiac tissue. The majority of these nitrated molecules are mitochondrial proteins with known functions in cellular energy metabolism and oxygen oxidation (26,27,29). However, whether the nitration of these mitochondrial proteins contributes to the development of diabetic cardiomyopathy remains largely unknown.The cysteine-rich protein metallothionein (MT) is a ubiquito...
Kupffer cells (KCs) are the resident macrophages of the liver, and they respond to and counteract metabolic stresses, such as those imposed by high-fat diet feeding in mouse models. However, little is known regarding the role of these cells in maintaining metabolic homeostasis under metabolically normal conditions. In this study, we found that depletion of KCs in vivo led to enhanced lipolysis in adipose tissue by increasing the expression of FGF21, a metabolic regulator, in hepatocytes. IL-1b secreted from KCs contributed to the suppression of FGF21 expression in hepatocytes. FGF21 overexpression led to a lean phenotype and enhanced lipolysis in mice. KC depletion resulted in a lack of IL-1b signaling in the liver, leading to elevated expression of FGF21 in hepatocytes. FGF21 promoted lipolysis in adipose tissue and led to hyperlipidemia and decreased body weight. The secretion of IL-1b in KCs was mediated by bacterial products. Antibiotic treatment also led to enhanced lipolysis. Therefore, the current study identified a physiological role of KCs in the regulation of adipose lipolysis.
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