eIn obesity, adipocyte hypertrophy and proinflammatory responses are closely associated with the development of insulin resistance in adipose tissue. However, it is largely unknown whether adipocyte hypertrophy per se might be sufficient to provoke insulin resistance in obese adipose tissue. Here, we demonstrate that lipid-overloaded hypertrophic adipocytes are insulin resistant independent of adipocyte inflammation. Treatment with saturated or monounsaturated fatty acids resulted in adipocyte hypertrophy, but proinflammatory responses were observed only in adipocytes treated with saturated fatty acids. Regardless of adipocyte inflammation, hypertrophic adipocytes with large and unilocular lipid droplets exhibited impaired insulin-dependent glucose uptake, associated with defects in GLUT4 trafficking to the plasma membrane. Moreover, Toll-like receptor 4 mutant mice (C3H/HeJ) with high-fat-diet-induced obesity were not protected against insulin resistance, although they were resistant to adipose tissue inflammation. Together, our in vitro and in vivo data suggest that adipocyte hypertrophy alone may be crucial in causing insulin resistance in obesity.

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Many mammalian cells release extracellular vesicles (EV) into circulation. These vesicles differ in ontogeny, size, and molecular composition. 1-5 The two most abundant fractions of EV are termed microvesicles (MV; 200-1000 nm), formed by budding of the cell membrane, and exosomes (EX; 50-200 nm), derived from multivesicular bodies. The last few years have seen intense interest in these vesicles given their biological role in cancer 6-9 and potential for diagnostic and therapeutic purposes. 10-13 To date, vesicles are often differentiated by size and density criteria 14-16 and the presence

Blocking phosphorylation of peroxisome proliferator–activated receptor (PPAR)γ at Ser273 is one of the key mechanisms for antidiabetes drugs to target PPARγ. Using high-throughput phosphorylation screening, we here describe that Gleevec blocks cyclin-dependent kinase 5–mediated PPARγ phosphorylation devoid of classical agonism as a PPARγ antagonist ligand. In high fat–fed mice, Gleevec improved insulin sensitivity without causing severe side effects associated with other PPARγ-targeting drugs. Furthermore, Gleevec reduces lipogenic and gluconeogenic gene expression in liver and ameliorates inflammation in adipose tissues. Interestingly, Gleevec increases browning of white adipose tissue and energy expenditure. Taken together, the results indicate that Gleevec exhibits greater beneficial effects on both glucose/lipid metabolism and energy homeostasis by blocking PPARγ phosphorylation. These data illustrate that Gleevec could be a novel therapeutic agent for use in insulin resistance and type 2 diabetes.

Recently, the interest in natural products for the treatment of cancer is increasing because they are the pre-screened candidates. In the present study, we demonstrate the therapeutic effect of celastrol, a triterpene extracted from the root bark of Chinese medicine on gastric cancer. The proliferation of AGS and YCC-2 cells were most sensitively decreased in six kinds of gastric cancer cell lines after the treatment with celastrol. Celastrol inhibited the cell migration and increased G1 arrest in cell-cycle populations in both cell lines. The treatment with celastrol significantly induced autophagy and apoptosis and increased the expression of autophagy and apoptosis-related proteins. We also found an increase in phosphorylated AMPK following a decrease in all phosphorylated forms of AKT, mTOR and S6K after the treatment with celastrol. Moreover, gastric tumor burdens were reduced in a dose-dependent manner by celastrol administration in a xenografted mice model. Taken together, celastrol distinctly inhibits the gastric cancer cell proliferation and induces autophagy and apoptosis. [BMB Reports 2014; 47(12): 697-702]

Extracellular vesicles (EVs) are important disease biomarkers; [1] they are ubiquitously present in bodily fluids and carry molecular cargo from their respective parental cells (e.g., transmembrane and intracellular proteins, mRNA, DNA, and microRNA). [2] EVs thus can serve as a blood-based analytical method to obtain and monitor diseases' molecular traits, [3] promoting better-informed clinical decisions. Indeed, EVs were found superior to conventional protein markers for tumor detection. [4-6] Key mutations (e.g., EGFRvIII, IDH1R132H, EGFR T790M) have also been detected in EVs. [6,7] Essential to diagnostic EV analysis is the ability to perform a given molecular test in true EV fractions, rather than in contaminated mixtures. In other words, EVs exist in complex heterogeneous matrices (Figure 1a) and retrieving pure vesicle populations is a pivotal first assay step. Among various EV isolation strategies, [1] size-exclusion chromatography (SEC) is increasingly adopted as a preferable isolation method for clinical samples, considering its low cost, much faster turnaround time compared to ultracentrifugation, and ease-of-operation. [8] SEC separates analytes based on their differential retention time in porous gels. When applied to plasma or serum, SEC produces well-defined vesicle fractions with most soluble proteins removed. [9] The current approaches, however, often fail to differentiate EVs from certain types of lipoprotein particles (LPPs), predominantly (very) low-density lipoproteins (V)LDL, due to size overlap. [10] With LPPs (≈10 15 particles mL −1) [11] significantly outnumbering EVs (≈10 7-10 9 particles mL −1 in healthy individuals), [12] SEC-prepared samples are susceptible to artifacts, including overestimation of actual EV counts, steric hindrance in immunoassays, and increased biological noise. This is especially important in the discovery phase of EV protein biomarkers, as the abundance of LPPs can confound the search for less-abundant EV-associated proteins. Density-gradient ultracentrifugation can be incorporated before or after SEC to separate EVs from LPPs but the combined process negates SEC's practical advantages. Here, we report a substantially improved chromatography method for rapid EV isolation from plasma samples. We noticed the contrast in surface charge properties between EVs, that are negatively charged, [13] and ApoB100-containing Purifying extracellular vesicles (EVs) from complex biological fluids is a critical step in analyzing EVs molecularly. Plasma lipoprotein particles (LPPs) are a significant confounding factor as they outnumber EVs >10 4-fold. Given their overlap in size, LPPs cannot be completely removed using standard sizeexclusion chromatography. Density-based separation of LPPs can be applied but is impractical for routine use in clinical research and practice. Here a new separation approach, known as dual-mode chromatography (DMC), capable of enriching plasma EVs, and depleting LPPs is reported. DMC conveniently integrates two orthogonal separation steps in a single co...

Highlights d Extracellular vesicles (EVs) are heterogeneous in their origins and contents d Expression of CD9 and CD81 are mutually exclusive at the single EV level d Cells shed fewer EVs during the process of immortalization and transformation d tRNA fragments are enriched in ribonucleoprotein complexes

We revealed the X-ray structure of PPARγ co-crystallized with SR1664 bound to the alternate binding site of PPARγ and confirmed that this blocks the phosphorylation of Ser273.