Siah2 protein mediates early events in commitment to an adipogenic pathway

© 2016 by The American Society for Biochemistry and Molecular Biology, Inc. Adipose tissue expansion occurs by increasing the size of existing adipocytes or by increasing the number of adipocytes via adipogenesis. Adipose tissue dysfunction in obesity is associated with adipocyte hypertrophy and impaired adipogenesis. We recently demonstrated that deletion of the ubiquitin ligase Siah2 is associated with enlarged adipocytes in lean or obese mice. In this study, we find that adipogenesis is impaired in 3T3-L1 preadipocytes stably transfected with Siah2 shRNA and that overexpression of Siah2 in non-precursor fibroblasts promotes adipogenesis. In the 3T3-L1 model, loss of Siah2 is associated with sustained β-catenin expression post-induction, but depletion of β-catenin only partially restores PPARγ expression and adipocyte formation. Using wild-type and Siah2-/- adipose tissue and adipose stromal vascular cells, we observe that Siah2 influences the expression of several factors that control adipogenesis, including Wnt pathway genes, β-catenin, Zfp432, and Bmp-4. Consistent with increased β-catenin levels in shSiah2 preadipocytes, Wnt10b is elevated in Siah2-/- adipose tissue and remains elevated in Siah2-/- primary stromal cells after addition of the induction mixture. However, addition of BMP-4 to Siah2-/- stromal cells reduces Wnt10b expression, reduces Zfp521 protein levels, and increases expression of Zfp423, a transcriptional regulator of peroxisome proliferator-activated receptor γ expression that controls commitment to adipogenesis and is repressed by Zfp521. These results indicate that Siah2 acts upstream of BMP-4 to regulate factors that control the commitment of adipocyte progenitors to an adipogenic pathway. Our findings reveal an essential role for Siah2 in the early events that signal undifferentiated progenitor cells to become mature adipocytes

The katanin microtubule severing protein in plants

Katanin is a heterodimeric microtubule (MT) severing protein that uses energy from ATP hydrolysis to generate internal breaks along MTs. Katanin p60, one of the two subunits, possesses ATPase and MT-binding/ severing activities, and the p 80 subunit is responsible for targeting of katanin to certain subcellular locations. In animals, katanin plays an important role in the release of MTs from their nucleation sites in the centrosome. It is also involved in severing MTs into smaller fragments which can serve as templates for further polymerization to increase MT number during meiotic and mitotic spindle assembly. Katanin homologs are present in a wide variety of plant species. The Arabidopsis katanin homolog has been shown to possess ATP-dependent MT severing activity in vitro and exhibit a punctate localization pattern at the cell cortex and the perinuclear region. Disruption of katanin functions by genetic mutations causes a delay in the disappearance of the perinuclear MT array and results in an aberrant organization of cortical MTs in elongating cells. Consequently, katanin mutations lead to defects in cell elongation, cellulose microfibril deposition, and hormonal responses. Studies of katanin in plants provide new insights into our understanding of its roles in cellular functions. © 2007 Institute of Botany, the Chinese Academy of Sciences

Up-regulation of P-glycoprotein reduces intracellular accumulation of beta amyloid: Investigation of P-glycoprotein as a novel therapeutic target for Alzheimer\u27s disease

Objectives: Several studies have suggested the efflux transporter P-glycoprotein (P-gp) to play a role in the etiology of Alzheimer\u27s disease through the clearance of amyloid beta (Aβ) from the brain. In this study, we aimed to investigate the possibility of P-gp as a potential therapeutic target for Alzheimer\u27s disease by examining the impact of P-gp up-regulation on the clearance of Aβ, a neuropathological hallmark of Alzheimer\u27s disease. Methods: Uptake studies for 125I-radiolabelled Aβ 1-40, and fluorescent immunostaining technique for P-gp and fluorescent imaging of Aβ 1-40 were carried out in LS-180 cells following treatment with drugs known to induce P-gp expression. Key findings: Approximately 10-35% decrease in 125I-Aβ 1-40 intracellular accumulation was observed in cells treated with rifampicin, dexamethasone, caffeine, verapamil, hyperforin, β-estradiol and pentylenetetrazole compared with control. Also, fluorescent micrographs showed an inverse relationship between levels of P-gp expression and 5-carboxyfluorescein labelled Aβ (FAM-Aβ 1-40) intracellular accumulation. Quantitative analysis of the micrographs revealed that the results were consistent with those of the uptake studies using 125I-Aβ 1-40. Conclusions: The investigated drugs were able to improve the efflux of Aβ 1-40 from the cells via P-gp up-regulation compared with control. Our results elucidate the importance of targeting Aβ clearance via P-gp up-regulation, which will be effective in slowing or halting the progression of Alzheimer\u27s disease. © 2011 The Authors JPP © 2011 Royal Pharmaceutical Society

Disruption of cortical microtubules by overexpression of green fluorescent protein-tagged α-tubulin 6 causes a marked reduction in cell wall synthesis

It has been known that the transverse orientation of cortical microtubules (MTs) along the elongation axis is essential for normal cell morphogenesis, but whether cortical MTs are essential for normal cell wall synthesis is still not clear. In the present study, we have investigated whether cortical MTs affect cell wall synthesis by direct alteration of the cortical MT organization in Arabidopsis thaliana. Disruption of the cortical MT organization by expression of an excess amount of green fluorescent protein-tagged α-tubulin 6 (GFP-TUA6) in transgenic Arabidopsis plants was found to cause a marked reduction in cell wall thickness and a decrease in the cell wall sugars glucose and xylose. Concomitantly, the stem strength of the GFP-TUA6 overexpressors was markedly reduced compared with the wild type. In addition, expression of excess GFP-TUA6 results in an alteration in cell morphogenesis and a severe effect on plant growth and development. Together, these results suggest that the proper organization of cortical MTs is essential for the normal synthesis of plant cell walls. © 2006 Institute of Botany, Chinese Academy of Sciences

EGF-stimulation activates the nuclear localization signal of SHP-1

Protein tyrosine phosphatase SHP-1 plays a critical role in the regulation of a variety of intracellular signaling pathways. SHP-1 is predominantly expressed in the cells of hematopoietic origin, and is recognized as a negative regulator of lymphocyte development and activation. SHP-1 consists of two Src homology 2 (SH2) domains and one protein tyrosine phosphatase (PTP) domain followed by a highly basic C-terminal tail containing tyrosyl phosphorylation sites. It is unclear how the C-terminal tail regulates SHP-1 function. We report the examination of the subcellular localization of a variety of truncated or mutated SHP-1 proteins fused with enhanced green fluorescent protein (EGFP) protein at either the N-terminal or the C-terminal end in different cell lines. Our data demonstrate that a nuclear localization signal (NLS) is located in the C-terminal tail of SHP-1 and the signal is primarily defined by three amino-acid residues (KRK) at the C-terminus. This signal is generally blocked in the native protein and can be exposed by fusing EGFP at the appropriate position or by domain truncation. We have also revealed that this NLS of SHP-1 is triggered by epidermal growth factor (EGF) stimulation and mediates translocation of SHP-1 from the cytosol to the nucleus in COS7 cell lines. These results not only demonstrate the importance of the C-terminal tail of SHP-1 in the regulation of nuclear localization, but also provide insights into its role in SHP-1-involved signal transduction pathways. © 2004 Wiley-Liss, Inc

Profluorogenic reductase substrate for rapid, selective, and sensitive visualization and detection of human cancer cells that overexpress NQO1

Achieving the vision of identifying and quantifying cancer-related events and targets for future personalized oncology is predicated on the existence of synthetically accessible and economically viable probe molecules fully able to report the presence of these events and targets in a rapid and highly selective and sensitive fashion. Delineated here are the design and evaluation of a newly synthesized turn-on probe whose intense fluorescent reporter signature is revealed only through probe activation by a specific intracellular enzyme present in tumor cells of multiple origins. Quenching of molecular probe fluorescence is achieved through unique photoinduced electron transfer between the naphthalimide dye reporter and a covalently attached, quinone-based enzyme substrate. Fluorescence of the reporter dye is turned on by rapid removal of the quinone quencher, an event that immediately occurs only after highly selective, two-electron reduction of the sterically and conformationally restricted quinone substrate by the cancer-associated human NAD(P)H:quinone oxidoreductase isozyme 1 (hNQO1). Successes of the approach include rapid differentiation of NQO1-expressing and -nonexpressing cancer cell lines via the unaided eye, flow cytometry, fluorescence imaging, and two-photon microscopy. The potential for use of the turn-on probe in longer-term cellular studies is indicated by its lack of influence on cell viability and its in vitro stability. © 2012 American Chemical Society

Cellular and molecular remodeling of inguinal adipose tissue mitochondria by dietary methionine restriction

© 2015 Elsevier Inc. Dietary methionine restriction (MR) produces a coordinated series of biochemical and physiological responses that improve biomarkers of metabolic health, increase energy expenditure, limit fat accretion and improve overall insulin sensitivity. Inguinal white adipose tissue (IWAT) is a primary target and site of action where the diet initiates transcriptional programs linked to enhancing both synthesis and oxidation of lipid. Using a combination of ex vivo approaches to assess dietary effects on cell morphology and function, we report that dietary MR produced a fourfold increase in multilocular, UCP1-expressing cells within this depot in conjunction with significant increases in mitochondrial content, size and cristae density. Dietary MR increased expression of multiple enzymes within the citric acid cycle, as well as respiratory complexes I, II and III. The physiological significance of these responses, evaluated in isolated mitochondria by high-resolution respirometry, was a significant increase in respiratory capacity measured using multiple substrates. The morphological, transcriptional and biochemical remodeling of IWAT mitochondria enhances the synthetic and oxidative capacity of this tissue and collectively underlies its expanded role as a significant contributor to the overall increase in metabolic flexibility and uncoupled respiration produced by the diet

FGF21 mediates the thermogenic and insulin-sensitizing effects of dietary methionine restriction but not its effects on hepatic lipid metabolism

© 2017 by the American Diabetes Association. Dietary methionine restriction (MR) produces a rapid and persistent remodeling of white adipose tissue (WAT), an increase in energy expenditure (EE), and enhancement of insulin sensitivity. Recent work established that hepatic expression of FGF21 is robustly increased by MR. Fgf212/2 mice were used to test whether FGF21 is an essential mediator of the physiological effects of dietary MR. The MR-induced increase in energy intake and EE and activation of thermogenesis in WAT and brown adipose tissue were lost in Fgf212/2 mice. However, dietary MR produced a comparable reduction in body weight and adiposity in both genotypes because of a negative effect of MR on energy intake in Fgf212/2 mice. Despite the similar loss in weight, dietary MR produced a more significant increase in in vivo insulin sensitivity in wild-Type than in Fgf212/2 mice, particularly in heart and inguinal WAT. In contrast, the ability of MR to regulate lipogenic and integrated stress response genes in liver was not compromised in Fgf212/2 mice. Collectively, these findings illustrate that FGF21 is a critical mediator of the effects of dietary MR on EE, remodeling of WAT, and increased insulin sensitivity but not of its effects on hepatic gene expression

The ubiquitin ligase Siah2 regulates obesity-induced adipose tissue inflammation

© 2015 The Obesity Society. Objective Chronic, low-grade adipose tissue inflammation associated with adipocyte hypertrophy is an important link in the relationship between obesity and insulin resistance. Although ubiquitin ligases regulate inflammatory processes, the role of these enzymes in metabolically driven adipose tissue inflammation is relatively unexplored. Herein, the effect of the ubiquitin ligase Siah2 on obesity-related adipose tissue inflammation was examined. Methods Wild-type and Siah2KO mice were fed a low- or high-fat diet for 16 weeks. Indirect calorimetry, body composition, and glucose and insulin tolerance were assayed along with glucose and insulin levels. Gene and protein expression, immunohistochemistry, adipocyte size distribution, and lipolysis were also analyzed. Results Enlarged adipocytes in obese Siah2KO mice were not associated with obesity-induced insulin resistance. Proinflammatory gene expression, stress kinase signaling, fibrosis, and crown-like structures were reduced in the Siah2KO adipose tissue, and Siah2KO adipocytes were more responsive to insulin-dependent inhibition of lipolysis. Loss of Siah2 increased expression of PPARγ target genes involved in lipid metabolism and decreased expression of proinflammatory adipokines regulated by PPARγ. Conclusions Siah2 links adipocyte hypertrophy with adipocyte dysfunction and recruitment of proinflammatory immune cells to adipose tissue. Selective regulation of PPARγ activity is a Siah2-mediated mechanism contributing to obesity-induced adipose tissue inflammation