No abstract

Adipocyte differentiation is a key process implicated in the pathogenesis of obesity and insulin resistance. Its regulation is triggered by a cascade of transcription factors, including the CCAAT/enhancer binding proteins (C/EBPs) and peroxisome proliferator-activated receptor-γ (PPARγ). Growth factors such as transforming growth factor-β1 (TGF-β1) are known to inhibit adipocyte differentiation in vitro, via the C/EBP pathway, and in vivo, but whether a downstream mediator of TGF-β1, connective tissue growth factor (CTGF), also known as CCN2, has a similar role is unknown. Mouse 3T3-L1 cells were differentiated into adipocytes by using standard methods, and effects and regulation of CTGF were studied. Intervention with recombinant human CTGF during differing stages of differentiation caused an inhibition in the development of the adipocyte phenotype, according to the gene expression of the differentiation markers adiponectin and PPARγ, as well as suppression of lipid accumulation and expression of the lipogenic enzyme glycerol-3-phosphate dehydrogenase. Whereas CTGF gene expression promptly fell by 90% as 3T3-L1 preadipocytes differentiated into mature adipocytes, CTGF mRNA expression was induced by added TGF-β1. CTGF applied to cells early in the course of differentiation inhibited total cell protein levels and nuclear localization of the β-isoform of C/EBP (C/EBP-β) and, subsequently, total cell C/EBP-α levels. CTGF also inhibited the adipocyte differentiation program in primary cultures of mouse preadipocytes. Expression of CTGF mRNA was twofold higher in the central fat depots of mice compared with subcutaneous fat, suggesting a potential role for CTGF in vivo. In summary, these data show that CTGF inhibits the adipocyte differentiation program.

Structural characterization of proteins in the gas phase is becoming increasingly popular, highlighting the need for a greater understanding of how proteins behave in the absence of solvent. It is clear that charged residues exert significant influence over structures in the gas phase due to strong Coulombic and hydrogen-bonding interactions. The net charge for a gaseous ion is easily identified by mass spectrometry, but the presence of zwitterionic pairs or salt bridges has previously been more difficult to detect. We show that these sites can be revealed by photoinduced electron transfer dissociation, which produces characteristic c and z ions only if zwitterionic species are present. Although previous work on small molecules has shown that zwitterionic pairs are rarely stable in the gas phase, we now demonstrate that charge-separated states are favored in larger molecules. Indeed, we have detected zwitterionic pairs in peptides and proteins where the net charge equals the number of basic sites, requiring additional protonation at nonbasic residues. For example, the small protein ubiquitin can sustain a zwitterionic conformer for all charge states up to 14+, despite having only 13 basic sites. Virtually all of the peptides/proteins examined herein contain zwitterionic sites if both acidic and basic residues are present and the overall charge density is low. This bias in favor of charge-separated states has important consequences for efforts to model gaseous proteins via computational analysis, which should consider not only charge state isomers that include salt bridges but also protonation at nonbasic residues.

Foot ulcers and poor wound healing are problematic for patients with diabetes. The beehive protectant Propolis can improve wound healing but whether it can improve healing in diabetic wounds has not been investigated. In this study, the effect of a single application of Propolis on epithelial closure, wound morphology, cellular infiltrate, and blood vessel density were investigated. Diabetes was induced in rats using streptozocin. After 6 weeks, diabetic and control animals were wounded and the wounds were treated with Propolis or saline as control. At days 6 and 12 animals were sacrificed and wounds were excised. Compared with controls, diabetes decreased epithelial closure and reepithelialization but had no effect on wound contraction. These delays were prevented by Propolis. At day 12, the impaired macrophage infiltration (C:1.49+/-0.09 vs. D:0.25+/-0.14), persistent neutrophil infiltration (C:0.22+/-0.19 vs. D:1.33+/-0.81), and increased myeloperoxidase activity (fourfold) in diabetic wounds were prevented by Propolis. Diabetes had no effect on wound volume, vessel number, or branch points. These novel data indicate that Propolis can accelerate wound healing in diabetes. As neutrophil infiltration is normalized, its mechanism of action may be through anti-inflammatory pathways. This result and the established safety profile of Propolis provide a rationale for studying topical application of this agent in a clinical setting.

Mesangial cell-derived factors alter monocyte activation and function through inflammatory pathways: possible pathogenic role in diabetic nephropathy. Am J Physiol Renal Physiol 297: F1229 -F1237, 2009. First published September 9, 2009 doi:10.1152/ajprenal.00074.2009.-Infiltration of macrophages to the kidney is a feature of early diabetic nephropathy. For this to happen monocytes must become activated, migrate from the circulation, and infiltrate the mesangium. This process involves degradation of extracellular matrix, a process mediated by matrix metalloproteinases (MMPs). In the present study we investigate the expression of proinflammatory cytokines TNF-␣, IL-6, and MMP-9 in glomeruli of control and diabetic rodents and use an in vitro coculture system to examine whether factors secreted by mesangial cells in response to a diabetic milieu can induce monocyte MMP-9 expression and infiltration. After 8 wk of diabetes, the glomerular level of TNF-␣, IL-6, and macrophage number and colocalization of MMP-9 with macrophage were increased (P Ͻ 0.01). Coculture of THP1 monocytes and glomerular mesangial cells in 5 or 25 mM glucose increased MMP-9 (5 mM: 65% and 25 mM: 112%; P Ͻ 0.05) and conditioned media degradative activity (5 mM: 30.0% and 25 mM: 33.5%: P Ͻ 0.05). These effects were reproduced by addition of mesangial cell conditioned medium to THP1 cells. High glucose (25 mM) increased TNF-␣, IL-6, and monocyte chemoattractant protein-1 in mesangial cell conditioned medium. These cytokines all increased adhesion and differentiation of THP1 cells (P Ͻ 0.05), but only TNF-␣ and IL-6 increased MMP-9 expression (50-and 60-fold, respectively; P Ͻ 0.05). Our results show that mesangial cellsecreted factors increase monocyte adhesion, differentiation, MMP expression, and degradative capacity. High glucose could augment these effects by increasing mesangial cell proinflammatory cytokine secretion. This mesangial cell-monocyte interaction may be important in activating monocytes to migrate from the circulation to the kidney in the early stages of diabetic nephropathy. matrix metalloproteinases; matrix degradation; cell adhesion and infiltration; inflammation response