Baek Soo Han

Metabolic Adaptation in Obesity and Type II Diabetes: Myokines, Adipokines and Hepatokines

Obesity and type II diabetes are characterized by insulin resistance in peripheral tissues. A high caloric intake combined with a sedentary lifestyle is the leading cause of these conditions. Whole-body insulin resistance and its improvement are the result of the combined actions of each insulin-sensitive organ. Among the fundamental molecular mechanisms by which each organ is able to communicate and engage in cross-talk are cytokines or peptides which stem from secretory organs. Recently, it was reported that several cytokines or peptides are secreted from muscle (myokines), adipose tissue (adipokines) and liver (hepatokines) in response to certain nutrition and/or physical activity conditions. Cytokines exert autocrine, paracrine or endocrine effects for the maintenance of energy homeostasis. The present review is focused on the relationship and cross-talk amongst muscle, adipose tissue and the liver as secretory organs in metabolic diseases.

A distinct death mechanism is induced by 1-methyl-4-phenylpyridinium or by 6-hydroxydopamine in cultured rat cortical neurons: Degradation and dephosphorylation of tau

We examined whether the well-known neurotoxins 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpyridinium ion (MPP(+)) recruit distinct cell death mechanisms using primary cultured neurons derived from day 16 embryonic rat cortices. Electron microscopy revealed that cell death induced by both 6-OHDA and MPP(+) was typified by a condensation of chromatin while prominent mitochondrial swelling was observed only in those cells treated with MPP(+). Co-treatment of cells with a pan-caspase inhibitor, Z-VAD-fmk, attenuated 6-OHDA-induced chromatin condensation and neuronal death. Co-treatment with such antioxidants as N-acetylcysteine or Mn-TBAP also suppressed 6-OHDA-induced cell death. None of these treatments attenuated MPP(+)-induced cell death although caspase inhibition abolished MPP(+)-induced chromatin condensation. Interestingly, in these paradigms of cell death, the N-terminus of tau was specifically cleaved and the levels of phosphorylated tau were markedly decreased following 6-OHDA treatment. By contrast, the C-terminus of tau was cleaved in MPP(+)-induced cell death while the levels of phosphorylated tau remained largely unaltered. Taken together, our results indicate that distinct cellular mechanisms appear to underlie neurotoxin-induced cortical neuronal cell death.

Identification of DNA Aptamers toward Epithelial Cell Adhesion Molecule via Cell-SELEX

The epithelial cell adhesion molecule (EpCAM, also known as CD326) is a transmembrane glycoprotein that is specifically detected in most adenocarcinomas and cancer stem cells. In this study, we performed a Cell systematic evolution of ligands by exponential enrichment (SELEX) experiment to isolate the aptamers against EpCAM. After seven round of Cell SELEX, we identified several aptamer candidates. Among the selected aptamers, EP166 specifically binds to cells expressing EpCAM with an equilibrium dissociation constant (Kd) in a micromolar range. On the other hand, it did not bind to negative control cells. Moreover, EP166 binds to J1ES cells, a mouse embryonic stem cell line. Therefore, the isolated aptamers against EpCAM could be used as a stem cell marker or in other applications in both stem cell and cancer studies.

Monitoring the Differentiation and Migration Patterns of Neural Cells Derived from Human Embryonic Stem Cells Using a Microfluidic Culture System

Microfluidics can provide unique experimental tools to visualize the development of neural structures within a microscale device, which is followed by guidance of neurite growth in the axonal isolation compartment. We utilized microfluidics technology to monitor the differentiation and migration of neural cells derived from human embryonic stem cells (hESCs). We co-cultured hESCs with PA6 stromal cells, and isolated neural rosette-like structures, which subsequently formed neurospheres in suspension culture. Tuj1-positive neural cells, but not nestin-positive neural precursor cells (NPCs), were able to enter the microfluidics grooves (microchannels), suggesting that neural cell-migratory capacity was dependent upon neuronal differentiation stage. We also showed that bundles of axons formed and extended into the microchannels. Taken together, these results demonstrated that microfluidics technology can provide useful tools to study neurite outgrowth and axon guidance of neural cells, which are derived from human embryonic stem cells.

Investigation of adipocyte proteome during the differentiation of brown preadipocytes.

UNLABELLED Brown adipocytes oxidize fatty acids to produce heat in response to cold or caloric overfeeding. The motivation and function of the development of brown fat may thus counteract obesity, though this remains uncertain. We investigated the brown adipocyte proteome by two-dimensional gel electrophoresis followed by mass spectrometry. Comparative analyses of proteins focused on total protein spots to filter differentially expressed proteins during the differentiation of mouse primary brown preadipocytes. A Western blot analysis was performed to verify the target proteins. The results indicated that 10 protein spots were differentially expressed with significant changes, including the three up-regulated proteins of prohibitin, hypoxanthine-guanine phosphoribosyltransferase, and enoyl-CoA hydratase protein; the 5 down-regulated proteins of triosephosphate isomerase, elongation factor 2, α-tropomyosin slow, endophilin-B1, and cofilin-1 (CFL1); and the two unequivocally expressed proteins of peroxiredoxin-1 and collagen α-1(i) chain precursor. We found that during brown adipogenesis, CFL1 has an inhibitory effect on brown adipocyte differentiation. The overexpression of CFL1 inhibited the brown fat deposition and repressed the brown marker genes UCP1, PRDM16, PGC-1α and PPARγ via actin dynamics and polymerization. These observations may be novel findings that bring new insight into the detailed mechanisms of brown adipogenesis and identify possible therapeutic targets for anti-obesity. BIOLOGICAL SIGNIFICANCE We use 2-DE to compare the proteomes of adipocytes during the brown adipogenesis of primary mouse preadipocytes. We identified 10 proteins that are differentially expressed. Among these, we found that the actin binding protein CFL1 inhibits the differentiation of brown preadipocytes. CFL1 overexpressing cells showed lower deposition of brown fat droplets, and the brown marker genes of UCP1, PRDM16, PGC-1α and PPARγ were decreased through actin dynamics and polymerization.

Recent Advances in Proteomic Studies of Adipose Tissues and Adipocytes

Obesity is a chronic disease that is associated with significantly increased levels of risk of a number of metabolic disorders. Despite these enhanced health risks, the worldwide prevalence of obesity has increased dramatically over the past few decades. Obesity is caused by the accumulation of an abnormal amount of body fat in adipose tissue, which is composed mostly of adipocytes. Thus, a deeper understanding of the regulation mechanism of adipose tissue and/or adipocytes can provide a clue for overcoming obesity-related metabolic diseases. In this review, we describe recent advances in the study of adipose tissue and/or adipocytes, focusing on proteomic approaches. In addition, we suggest future research directions for proteomic studies which may lead to novel treatments of obesity and obesity-related diseases.

Selection of Aptamers for Mature White Adipocytes by Cell SELEX Using Flow Cytometry

Background Adipose tissue, mainly composed of adipocytes, plays an important role in metabolism by regulating energy homeostasis. Obesity is primarily caused by an abundance of adipose tissue. Therefore, specific targeting of adipose tissue is critical during the treatment of obesity, and plays a major role in overcoming it. However, the knowledge of cell-surface markers specific to adipocytes is limited. Methods and Results We applied the CELL SELEX (Systematic Evolution of Ligands by EXponential enrichment) method using flow cytometry to isolate molecular probes for specific recognition of adipocytes. The aptamer library, a mixture of FITC-tagged single-stranded random DNAs, is used as a source for acquiring molecular probes. With the increasing number of selection cycles, there was a steady increase in the fluorescence intensity toward mature adipocytes. Through 12 rounds of SELEX, enriched aptamers showing specific recognition toward mature 3T3-L1 adipocyte cells were isolated. Among these, two aptamers (MA-33 and 91) were able to selectively bind to mature adipocytes with an equilibrium dissociation constant (Kd) in the nanomolar range. These aptamers did not bind to preadipocytes or other cell lines (such as HeLa, HEK-293, or C2C12 cells). Additionally, it was confirmed that MA-33 and 91 can distinguish between mature primary white and primary brown adipocytes. Conclusions These selected aptamers have the potential to be applied as markers for detecting mature white adipocytes and monitoring adipogenesis, and could emerge as an important tool in the treatment of obesity.

Graphene oxide induces apoptotic cell death in endothelial cells by activating autophagy via calcium-dependent phosphorylation of c-Jun N-terminal kinases.

Despite the rapid expansion of the biomedical applications of graphene oxide (GO), safety issues related to GO, particularly with regard to its effects on vascular endothelial cells (ECs), have been poorly evaluated. To explore possible GO-mediated vasculature cytotoxicity and determine lateral GO size relevance, we constructed four types of GO: micrometer-sized GO (MGO; 1089.9±135.3nm), submicrometer-sized GO (SGO; 390.2±51.4nm), nanometer-sized GO (NGO; 65.5±16.3nm), and graphene quantum dots (GQDs). All types but GQD showed a significant decrease in cellular viability in a dose-dependent manner. Notably, SGO or NGO, but not MGO, potently induced apoptosis while causing no detectable necrosis. Subsequently, SGO or NGO markedly induced autophagy through a process dependent on the c-Jun N-terminal kinase (JNK)-mediated phosphorylation of B-cell lymphoma 2 (Bcl-2), leading to the dissociation of Beclin-1 from the Beclin-1-Bcl-2 complex. Autophagy suppression attenuated the SGO- or NGO-induced apoptotic cell death of ECs, suggesting that SGO- or NGO-induced cytotoxicity is associated with autophagy. Moreover, SGO or NGO significantly induced increased intracellular calcium ion (Ca2+) levels. Intracellular Ca2+ chelation with BAPTA-AM significantly attenuated microtubule-associated protein 1A/1B-light chain 3-II accumulation and JNK phosphorylation, resulting in reduced autophagy. Furthermore, we found that SGO or NGO induced Ca2+ release from the endoplasmic reticulum through the PLC β3/IP3/IP3R signaling axis. These results elucidate the mechanism underlying the size-dependent cytotoxicity of GOs in the vasculature and may facilitate the development of a safer biomedical application of GOs. STATEMENT OF SIGNIFICANCE Graphene oxide (GO) have received considerable attention with respect to their utilization in biomedical applications. However, GO-related safety issues concerning human vasculature are very limited. In this manuscript, we report for the first time the differential size-related biological effects of GOs on endothelial cells (ECs). Notably, Subnanometer- and nanometersized GOs induce apoptotic death in ECs via autophagy activation. We propose a molecular mechanism for the GO-induced autophagic cell death through the PLCβ3/IP3/Ca2+/JNK signaling axis. Our findings could be provide a better understanding of the GO sizedependent cytotoxicity in vasculature and facilitate the future development of safer biomedical applications of GOs.

Translocation and oligomerization of Bax is regulated independently by activation of p38 MAPK and caspase-2 during MN9D dopaminergic neurodegeneration

Bax is translocated into the mitochondrial membrane and oligomerized therein to initiate mitochondrial apoptotic signaling. Our previous study indicated that reactive oxygen species (ROS)-mediated activation of mitogen-activated protein kinase (MAPK) and caspase is critically involved in 6-hydroxydopamine (6-OHDA)-mediated neurodegeneration. Here, we specifically attempted to examine whether and how these death signaling pathways may be linked to Bax translocation and oligomerization. We found that 6-OHDA treatment triggered translocation and oligomerization of Bax onto the mitochondria in MN9D dopaminergic neuronal cells. These events preceded cytochrome c release into the cytosol. Cross-linking assay revealed that co-treatment with a ROS scavenger or a pan-caspase inhibitor inhibited 6-OHDA-induced Bax oligomerization. Among several candidates of ROS-activated MAPKs and caspases, we found that co-treatment with PD169316 or VDVAD specifically inhibited 6-OHDA-induced Bax oligomerization, suggesting critical involvement of p38 MAPK and caspase-2. Consequently, overexpression of a dominant negative form of p38 MAPK or a shRNA-mediated knockdown of caspase-2 indeed inhibited 6-OHDA-induced Bax oligomerization. However, activation of p38 MAPK and caspase-2 was independently linked to oligomerization of Bax. This specificity was largely confirmed with a Bax 6A7 antibody known to detect activated forms of Bax on the mitochondria. Taken together, our data suggest that there is an independent amplification loop of Bax translocation and oligomerization via caspase-2 and p38 MAPK during ROS-mediated dopaminergic neurodegeneration.

Stimulation of angiogenesis and survival of endothelial cells by human monoclonal Tie2 receptor antibody.

Angiopoietin-1 (Ang1) and its endothelium-specific receptor, tyrosine kinase with Ig and epidermal growth factor homology domain 2 (Tie2), play critical roles in vascular development. Although the Ang1/Tie2 system has been considered a promising target for therapeutic neovascularization, several imitations of large-scale production have hampered the development of recombinant Ang1 for therapeutics. In this study, we produced a fully human agonistic antibody against Tie2, designated 1-4h, and tested the applicability of 1-4h as an alternative to native Ang1 in therapeutic angiogenesis. 1-4h significantly enhanced the phosphorylation of Tie2 in a dose- and time-dependent manner in human Tie2-expressing HEK293 cells and human umbilical vein endothelial cells. Moreover, 1-4h induced the activation of Tie2-mediated intracellular signaling such as AKT, eNOS, MAPK, and Focal Adhesion Kinase p125(FAK). In addition, 1-4h increased the chemotactic motility and capillary-like tube formation of endothelial cells in vitro and enhanced the survival of serum-deprived endothelial cells. Taken together, our data clearly suggest that a human Tie2 agonistic antibody is a potentially useful therapeutic approach for the treatment of several ischemic diseases including delayed-wound healing and ischemic heart and limb diseases.