Jong Hyuk Yoon

Comparative analysis of the secretory proteome of human adipose stromal vascular fraction cells during adipogenesis.

Adipogenesis is a complex process that is accompanied by a number of molecular events. In this study, a proteomic approach was adopted to identify secretory factors associated with adipogenesis. A label‐free shotgun proteomic strategy was implemented to analyze proteins secreted by human adipose stromal vascular fraction cells and differentiated adipocytes. A total of 474 proteins were finally identified and classified according to quantitative changes and statistical significances. Briefly, 177 proteins were significantly upregulated during adipogenesis (Class I), whereas 60 proteins were significantly downregulated (Class II). Changes in the expressions of several proteins were confirmed by quantitative RT‐PCR and immunoblotting. One obvious finding based on proteomic data was that the amounts of several extracellular modulators of Wnt and transforming growth factor‐β (TGF‐β) signaling changed during adipogenesis. The expressions of secreted frizzled‐related proteins, dickkopf‐related proteins, and latent TGF‐β‐binding proteins were found to be altered during adipogenesis, which suggests that they participate in the fine regulation of Wnt and TGF‐β signaling. This study provides useful tools and important clues regarding the roles of secretory factors during adipogenic differentiation, and provides information related to obesity and obesity‐related metabolic diseases.

Comparative proteomic analysis of the insulin-induced L6 myotube secretome

Emerging evidence has revealed an endocrine function for skeletal muscle; in fact, certain anti‐inflammatory cytokines are secreted only from contractile skeletal muscle. However, the skeletal muscle secretome as a whole is poorly characterized, as is how it changes in response to extracellular stimuli. Herein, we sought to identify and characterize the members of the skeletal muscle secretome, and to determine which protein secretion levels were modulated in response to insulin stimulation. To conduct these studies, we treated differentiated L6 rat skeletal muscle cells with insulin or left them untreated, and we comparatively analyzed the proteins secreted into the media. We fractionated this conditioned media using offline RP HPLC, digested the fractionated proteins, and analyzed the resulting peptides with LC‐ESI‐MS/MS. We identified a total of 254 proteins, and by using three different filtering methods, we identified 153 of these as secretory proteins. Fourteen proteins were secreted at higher levels under insulin stimulation, including several proteins known to be highly secreted in metabolic diseases; 19 proteins were secreted at lower levels under insulin stimulation. These result not only pinpointed several previously unknown, insulin induced, secretory proteins of skeletal muscle, it also described a novel approach for conditioned secretome analysis.

Lysophosphatidylcholine Activates Adipocyte Glucose Uptake and Lowers Blood Glucose Levels in Murine Models of Diabetes

Glucose homeostasis is maintained by the orchestration of peripheral glucose utilization and hepatic glucose production, mainly by insulin. In this study, we found by utilizing a combined parallel chromatography mass profiling approach that lysophosphatidylcholine (LPC) regulates glucose levels. LPC was found to stimulate glucose uptake in 3T3-L1 adipocytes dose- and time-dependently, and this activity was found to be sensitive to variations in acyl chain lengths and to polar head group types in LPC. Treatment with LPC resulted in a significant increase in the level of GLUT4 at the plasma membranes of 3T3-L1 adipocytes. Moreover, LPC did not affect IRS-1 and AKT2 phosphorylations, and LPC-induced glucose uptake was not influenced by pretreatment with the PI 3-kinase inhibitor LY294002. However, glucose uptake stimulation by LPC was abrogated both by rottlerin (a protein kinase Cδ inhibitor) and by the adenoviral expression of dominant negative protein kinase Cδ. In line with its determined cellular functions, LPC was found to lower blood glucose levels in normal mice. Furthermore, LPC improved blood glucose levels in mouse models of type 1 and 2 diabetes. These results suggest that an understanding of the mode of action of LPC may provide a new perspective of glucose homeostasis.

CXCL12 secreted from adipose tissue recruits macrophages and induces insulin resistance in mice

Aims/hypothesisObesity-induced inflammation is initiated by the recruitment of macrophages into adipose tissue. The recruited macrophages, called adipose tissue macrophages, secrete several proinflammatory cytokines that cause low-grade systemic inflammation and insulin resistance. The aim of this study was to find macrophage-recruiting factors that are thought to provide a crucial connection between obesity and insulin resistance.MethodsWe used chemotaxis assay, reverse phase HPLC and tandem MS analysis to find chemotactic factors from adipocytes. The expression of chemokines and macrophage markers was evaluated by quantitative RT-PCR, immunohistochemistry and FACS analysis.ResultsWe report our finding that the chemokine (C-X-C motif) ligand 12 (CXCL12, also known as stromal cell-derived factor 1), identified from 3T3-L1 adipocyte conditioned medium, induces monocyte migration via its receptor chemokine (C-X-C motif) receptor 4 (CXCR4). Diet-induced obese mice demonstrated a robust increase of CXCL12 expression in white adipose tissue (WAT). Treatment of obese mice with a CXCR4 antagonist reduced macrophage accumulation and production of proinflammatory cytokines in WAT, and improved systemic insulin sensitivity.Conclusions/interpretationIn this study we found that CXCL12 is an adipocyte-derived chemotactic factor that recruits macrophages, and that it is a required factor for the establishment of obesity-induced adipose tissue inflammation and systemic insulin resistance.

Time-resolved X-ray scattering and calorimetric studies on the crystallization behaviors of poly(ethylene terephthalate) (PET) and its copolymers containing isophthalate units

Abstract Time-resolved small-angle X-ray scattering (SAXS) measurements were carried out for PET and its copolymers undergoing isothermal crystallization. Wide-angle X-ray diffraction and differential scanning calorimetric measurements were also performed. Our data analysis of the SAXS results for PET and the copolymers clearly demonstrate that the one layer thickness l 1 (derived directly from the correlation functions of the measured SAXS profiles) is the lamellar crystal thickness d c , not the amorphous layer thickness d a . The observed d c values are found to be always smaller than d a , regardless of polymer composition. d c is highly dependent on the crystallization temperature, showing that the degree of supercooling is the major factor determining the thickness of lamellar crystals. No thickening, however, occurs in isothermal crystallizations. The kinked isophthalate units in the copolymer are found to be mostly excluded from the lamellar crystals during the crystallization process, leading to an increase of the amorphous layer thickness. Moreover, the kinked, rigid nature of the isophthalate unit was found to restrict crystal growth along the chain axis of the copolymers and also to lower their crystallinity. Unlike d c , d a decreases with crystallization time, causing a reduction of the long period in the lamellar stack. This drop in d a is interpreted in this paper by taking into account several factors that could influence crystallization behavior: the d a distribution in the lamellar stacks and its variation with time, the number of lamellae in the lamellar stacks and their effect on the SAXS profile, and the relaxation of polymer chains in the amorphous layers.

Emodin Regulates Glucose Utilization by Activating AMP-activated Protein Kinase

Background: AMPK activation improves glucose tolerance and insulin sensitivity. Results: Emodin increases glucose uptake in skeletal muscle and lowers blood glucose levels via AMPK activation. Conclusion: Administration of emodin leads to increased glucose tolerance and insulin sensitivity in vivo. Significance: Our results highlight the potential value of emodin as a drug for the treatment of diabetes. AMP-activated protein kinase has been described as a key signaling protein that can regulate energy homeostasis. Here, we aimed to characterize novel AMP-activated kinase (AMPK)-activating compounds that have a much lower effective concentration than metformin. As a result, emodin, a natural anthraquinone derivative, was shown to stimulate AMPK activity in skeletal muscle and liver cells. Emodin enhanced GLUT4 translocation and [14C]glucose uptake into the myotube in an AMPK-dependent manner. Also, emodin inhibited glucose production by suppressing the expression of key gluconeogenic genes, such as phosphoenolpyruvate carboxykinase and glucose-6-phosphatase, in hepatocytes. Furthermore, we found that emodin can activate AMPK by inhibiting mitochondrial respiratory complex I activity, leading to increased reactive oxygen species and Ca2+/calmodulin-dependent protein kinase kinase activity. Finally, we confirmed that a single dose administration of emodin significantly decreased the fasting plasma glucose levels and improved glucose tolerance in C57Bl/6J mice. Increased insulin sensitivity was also confirmed after daily injection of emodin for 8 days using an insulin tolerance test and insulin-stimulated PI3K phosphorylation in wild type and high fat diet-induced diabetic mouse models. Our study suggests that emodin regulates glucose homeostasis in vivo by AMPK activation and that this may represent a novel therapeutic principle in the treatment of type 2 diabetic models.

Proteomic analysis of tumor necrosis factor-alpha (TNF-α)-induced L6 myotube secretome reveals novel TNF-α-dependent myokines in diabetic skeletal muscle.

There is a strong possibility that skeletal muscle can respond to irregular metabolic states by secreting specific cytokines. Obesity-related chronic inflammation, mediated by pro-inflammatory cytokines, is believed to be one of the causes of insulin resistance that results in type 2 diabetes. Here, we attempted to identify and characterize the members of the skeletal muscle secretome in response to tumor necrosis factor-alpha (TNF-α)-induced insulin resistance. To conduct this study, we comparatively analyzed the media levels of proteins released from L6 skeletal muscle cells. We found 28 TNF-α modulated secretory proteins by using separate filtering methods: Gene Ontology, SignalP, and SecretomeP, as well as the normalized Spectral Index for label-free quantification. Ten of these secretory proteins were increased and 18 secretory proteins were decreased by TNF-α treatment. Using microarray analysis of Zuker diabetic rat skeletal muscle combined with bioinformatics and Q-PCR, we found a correlation between TNF-α-mediated insulin resistance and type 2 diabetes. This novel approach combining analysis of the conditioned secretome and transcriptome has identified several previously unknown, TNF-α-dependent secretory proteins, which establish a foothold for research on the different causes of insulin resistance and their relationships with each other.

Secretomics for skeletal muscle cells: a discovery of novel regulators?

Metabolic tissues, including skeletal muscle, adipose tissue and the digestive system, dynamically secrete various factors depending on the metabolic state, communicate with each other and orchestrate functions to maintain body homeostasis. Skeletal muscle secretes cytokines such as interleukin-6 (IL-6), IL-15, fibroblast growth factor-21 (FGF21) and IL-8. These compounds, myokines, play important roles in biological homeostasis such as energy metabolism, angiogenesis and myogenesis. New technological advances have allowed secretomics - analysis of the secretome - to be performed. The application of highly sensitive mass spectrometry makes qualitative and quantitative analysis of the secretome of skeletal muscle possible. Secretory proteins derived from skeletal muscle cells under various conditions were analyzed, and many important factors were suggested. In-depth studies of the secretome from metabolic cells in various conditions are strongly recommended. This study will provide information on methods of novel communication between metabolic tissues.

Melanocortins induce interleukin 6 gene expression and secretion through melanocortin receptors 2 and 5 in 3T3-L1 adipocytes

Interleukin 6 (IL6) is a pleiotropic cytokine that not only affects the immune system, but also plays an active role in many physiological events in various organs. Notably, 35% of systemic IL6 originates from adipose tissues under noninflammatory conditions. Here, we describe a previously unknown function of melanocortins in regulating Il6 gene expression and production in 3T3-L1 adipocytes through membrane receptors which are called melanocortin receptors (MCRs). Of the five MCRs that have been cloned, MC2R and MC5R are expressed during adipocyte differentiation. α-Melanocyte-stimulating hormone (α-MSH) or ACTH treatment of 3T3-L1 adipocytes induces Il6 gene expression and production in a time- and concentration-dependent manner via various signaling pathways including the protein kinase A, p38 mitogen-activated protein kinase, cJun N-terminal kinase, and IκB kinase pathways. Specific inhibition of MC2R and MC5R expression with short interfering Mc2r and Mc5r RNAs significantly attenuated the α-MSH-induced increase of intracellular cAMP and both the level of Il6 mRNA and secretion of IL6 in 3T3-L1 adipocytes. Finally, when injected into mouse tail vein, α-MSH dramatically increased the Il6 transcript levels in epididymal fat pads. These results suggest that α-MSH in addition to ACTH may function as a regulator of inflammation by regulating cytokine production.

Proteomic analysis of hypoxia-induced U373MG glioma secretome reveals novel hypoxia-dependent migration factors

High‐grade gliomas are one of the most common brain tumors and notorious for poor prognosis due to their malignant nature. Gliomas have an extensive area of hypoxia, which is critical for glioma progression by inducing aggressiveness and activating the angiogenesis process in the tumor microenvironment. To resolve the factors responsible for the highly malignant nature of gliomas, we comprehensively profiled the U373MG glioma cell secretome—exosome and soluble fraction under hypoxic and normoxic conditions. A total of 239 proteins were identified from the exosome and soluble fractions. Vascular endothelial growth factor, stanniocalcin 1 (STC1) and stanniocalcin 2, and insulin‐like growth factor binding protein 3 and 6, enriched in the soluble fraction, and lysyl oxidase homolog 2 enriched in the exosomal fraction were identified as upregulated proteins by hypoxia based on a label‐free quantitative analysis. STCs and insulin‐like growth factor binding proteins, which were identified as secretory proteins under hypoxic conditions, were highly correlated with glioma grade in human patients by microarray analysis. An in vitro scratch wound assay revealed that STC1 and 2 have important functions in the induction of cell migration in a hypoxia‐dependent manner, suggesting that they are hypoxia‐dependent migration factors.