Min Jeong Ryu

The Roles of Adipokines, Proinflammatory Cytokines, and Adipose Tissue Macrophages in Obesity-Associated Insulin Resistance in Modest Obesity and Early Metabolic Dysfunction

The roles of adipokines, proinflammatory cytokines, and adipose tissue macrophages in obesity-associated insulin resistance have been explored in both animal and human studies. However, our current understanding of obesity-associated insulin resistance relies on studies of artificial metabolic extremes. The purpose of this study was to explore the roles of adipokines, proinflammatory cytokines, and adipose tissue macrophages in human patients with modest obesity and early metabolic dysfunction. We obtained omental adipose tissue and fasting blood samples from 51 females undergoing gynecologic surgery. We investigated serum concentrations of proinflammatory cytokines and adipokines as well as the mRNA expression of proinflammatory and macrophage phenotype markers in visceral adipose tissue using ELISA and quantitative RT-PCR. We measured adipose tissue inflammation and macrophage infiltration using immunohistochemical analysis. Serum levels of adiponectin and leptin were significantly correlated with HOMA-IR and body mass index. The levels of expression of MCP-1 and TNF-α in visceral adipose tissue were also higher in the obese group (body mass index ≥ 25). The expression of mRNA MCP-1 in visceral adipose tissue was positively correlated with body mass index (r = 0.428, p = 0.037) but not with HOMA-IR, whereas TNF-α in visceral adipose tissue was correlated with HOMA-IR (r = 0.462, p = 0.035) but not with body mass index. There was no obvious change in macrophage phenotype or macrophage infiltration in patients with modest obesity or early metabolic dysfunction. Expression of mRNA CD163/CD68 was significantly related to mitochondrial-associated genes and serum inflammatory cytokine levels of resistin and leptin. These results suggest that changes in the production of inflammatory biomolecules precede increased immune cell infiltration and induction of a macrophage phenotype switch in visceral adipose tissue. Furthermore, serum resistin and leptin have specific roles in the regulation of adipose tissue macrophages in patients with modest obesity or early metabolic dysfunction.

Crif1 Deficiency Reduces Adipose OXPHOS Capacity and Triggers Inflammation and Insulin Resistance in Mice

Impaired mitochondrial oxidative phosphorylation (OXPHOS) has been proposed as an etiological mechanism underlying insulin resistance. However, the initiating organ of OXPHOS dysfunction during the development of systemic insulin resistance has yet to be identified. To determine whether adipose OXPHOS deficiency plays an etiological role in systemic insulin resistance, the metabolic phenotype of mice with OXPHOS–deficient adipose tissue was examined. Crif1 is a protein required for the intramitochondrial production of mtDNA–encoded OXPHOS subunits; therefore, Crif1 haploinsufficient deficiency in mice results in a mild, but specific, failure of OXPHOS capacity in vivo. Although adipose-specific Crif1-haploinsufficient mice showed normal growth and development, they became insulin-resistant. Crif1-silenced adipocytes showed higher expression of chemokines, the expression of which is dependent upon stress kinases and antioxidant. Accordingly, examination of adipose tissue from Crif1-haploinsufficient mice revealed increased secretion of MCP1 and TNFα, as well as marked infiltration by macrophages. These findings indicate that the OXPHOS status of adipose tissue determines its metabolic and inflammatory responses, and may cause systemic inflammation and insulin resistance.

Growth differentiation factor 15 is a myomitokine governing systemic energy homeostasis

Reduced mitochondrial electron transport chain activity promotes longevity and improves energy homeostasis via cell-autonomous and –non-autonomous factors in multiple model systems. This mitohormetic effect is thought to involve the mitochondrial unfolded protein response (UPRmt), an adaptive stress-response pathway activated by mitochondrial proteotoxic stress. Using mice with skeletal muscle–specific deficiency of Crif1 (muscle-specific knockout [MKO]), an integral protein of the large mitoribosomal subunit (39S), we identified growth differentiation factor 15 (GDF15) as a UPRmt-associated cell–non-autonomous myomitokine that regulates systemic energy homeostasis. MKO mice were protected against obesity and sensitized to insulin, an effect associated with elevated GDF15 secretion after UPRmt activation. In ob/ob mice, administration of recombinant GDF15 decreased body weight and improved insulin sensitivity, which was attributed to elevated oxidative metabolism and lipid mobilization in the liver, muscle, and adipose tissue. Thus, GDF15 is a potent mitohormetic signal that safeguards against the onset of obesity and insulin resistance.

Mitochondrial Localization and Regulation of BRAFV600E in Thyroid Cancer: A Clinically Used RAF Inhibitor Is Unable to Block the Mitochondrial Activities of BRAFV600E

CONTEXT The oncogenic BRAF(V600E) mutation results in an active structural conformation characterized by greatly elevated ERK activity. However, additional cellular effects caused by subcellular action of BRAF(V600E) remain to be identified. OBJECTIVE To explore these effects, differences in the subcellular localization of wild-type and mutant BRAF in thyroid cancer were investigated. RESULTS A significant proportion of endogenous and exogenous BRAF(V600E), but not wild-type BRAF, was detected in the mitochondrial fraction, similar to other BRAF mutants including BRAF(V600D), BRAF(V600K), BRAF(V600R), and BRAF(G469A), which showed elevated kinase activity and mitochondrial localization. Induced expression of BRAF(V600E) suppressed the apoptotic responses against staurosporine and TNFα/cycloheximide. Interestingly, the mitochondrial localization and antiapoptotic activities of BRAF(V600E) were unaffected by sorafenib and U0126 suppression of MAPK kinase (MEK) and ERK activities. Similarly, although the RAF inhibitor sorafenib effectively inhibited MEK/ERK activation, it did not block the mitochondrial localization of BRAF(V600E). In addition, inducible expression of BRAF(V600E) increased the glucose uptake rate and decreased O(2) consumption, suggesting that BRAF(V600E) reduces mitochondrial oxidative phosphorylation, a signature feature of cancer cells. Again, these metabolic alterations resulted by BRAF(V600E) expression were not affected by the treatment of thyroid cells by sorafenib. Therefore, RAF and MEK inhibitors are unable to block the antiapoptotic activity of BRAF(V600E) or correct the high glucose uptake rate and glycolytic activity and suppressed mitochondrial oxidative phosphorylation induced by BRAF(V600E). CONCLUSIONS The mitochondrial localization observed in oncogenic BRAF mutants might be related to their altered responses to apoptotic stimuli and characteristic metabolic phenotypes found in thyroid cancer. The inability of MEK and RAF inhibitors, U0126 and sorafenib, respectively, to block the mitochondrial localization of BRAF(V600E) has additional therapeutic implications for BRAF(V600E)-positive thyroid cancers.

Cross-Regulation between Oncogenic BRAFV600E Kinase and the MST1 Pathway in Papillary Thyroid Carcinoma

Background The BRAFV600E mutation leading to constitutive signaling of MEK-ERK pathways causes papillary thyroid cancer (PTC). Ras association domain family 1A (RASSF1A), which is an important regulator of MST1 tumor suppressor pathways, is inactivated by hypermethylation of its promoter region in 20 to 32% of PTC. However, in PTC without RASSF1A methylation, the regulatory mechanisms of RASSF1A-MST1 pathways remain to be elucidated, and the functional cooperation or cross regulation between BRAFV600E and MST1,which activates Foxo3,has not been investigated. Methodology/Principal Findings The negative regulators of the cell cycle, p21 and p27, are strongly induced by transcriptional activation of FoxO3 in BRAFV600E positive thyroid cancer cells. The FoxO3 transactivation is augmented by RASSF1A and the MST1 signaling pathway. Interestingly, introduction of BRAFV600Emarkedly abolished FoxO3 transactivation and resulted in the suppression of p21 and p27 expression. The suppression of FoxO3 transactivation by BRAFV600Eis strongly increased by coexpression of MST1 but it is not observed in the cells in which MST1, but not MST2,is silenced. Mechanistically, BRAFV600Ewas able to bind to the C-terminal region of MST1 and resulted in the suppression of MST1 kinase activities. The induction of the G1-checkpoint CDK inhibitors, p21 and p27,by the RASSF1A-MST1-FoxO3 pathway facilitates cellular apoptosis, whereasaddition of BRAFV600E inhibits the apoptotic processes through the inactivation of MST1. Transgenic induction of BRAFV600Ein the thyroid gland results in cancers resembling human papillary thyroid cancers. The development of BRAFV600Etransgenic mice with the MST1 knockout background showed that these mice had abundant foci of poorly differentiated carcinomas and large areas without follicular architecture or colloid formation. Conclusions/Significance The results of this study revealed that the oncogenic effect of BRAFV600E is associated with the inhibition of MST1 tumor suppressor pathways, and that the activity of RASSF1A-MST1-FoxO3 pathways determines the phenotypes of BRAFV600E tumors.

Dual specificity phosphatase 6 as a predictor of invasiveness in papillary thyroid cancer

OBJECTIVE The genetic mutations causing the constitutive activation of MEK/ERK have been regarded as an initiating factor in papillary thyroid carcinoma (PTC). The ERK-specific dual specificity phosphatase 6 (DUSP6) is part of the ERK-dependent transcriptional output. Therefore, the coordinated regulation of the activities of ERK kinases and DUSP6 may need to be reestablished to make new balances in PTC. METHODS To investigate the role of DUSP6 in the regulation of ERK1/2 (MAPK3/1)-dependent transcription, 42 benign neoplasms and 167 PTCs were retrospectively analyzed by immunohistochemistry with dideoxy sequencing to detect BRAF(V600E) mutation. RESULTS The expressions of total ERK1/2, DUSP6, c-Fos (FOS), c-Myc (MYC), cyclin D1, and PCNA were markedly increased in PTC compared with those in benign neoplasms. However, phospho-ERK1/2 was detected in only eight (4.8%) cases out of 167 PTC samples. Unexpectedly, the staining intensity and nuclear localization of ERK1/2 were not affected by the presence or absence of the BRAF(V600E) mutation. However, the expressions of c-Fos and PCNA were elevated in BRAF(V600E)-positive PTC compared with those in BRAF(V600E)-negative PTC. Interestingly, the higher staining intensities of DUSP6 were associated with the level of total ERK1/2 expression (P=0.04) and with high-risk biological features such as age (P=0.05), tumor size (P=0.01), and extrathyroidal extension (linear by linear association, P=0.02). In addition, DUSP6 silencing significantly decreased the cell viability and migration rate of FRO cells. CONCLUSIONS The coordinated upregulation of total ERK1/2 and its phosphatase, DUSP6, is related to bare detection of phospho-ERK1/2 in PTC regardless of BRAF(V)(600E) mutation status. A link between DUSP6 expression and high-risk features of PTC suggested that DUSP6 is an important independent factor affecting the signaling pathways in established PTC.

The indole derivative NecroX‐7 improves nonalcoholic steatohepatitis in ob/ob mice through suppression of mitochondrial ROS/RNS and inflammation

Nonalcoholic steatohepatitis (NASH) is associated with cirrhosis and hepatocellular carcinoma. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) play key roles in the development of the disease. However, the therapeutic target of NASH has not been fully defined and new treatments are needed. We investigated the protective effects of the antioxidant indole‐derived NecroX‐7 in a NASH mouse model using leptin‐deficient ob/ob and methionine‐ and choline‐deficient (MCD) diet‐fed ob/ob mice.

Aberrant L1 Cell Adhesion Molecule Affects Tumor Behavior and Chemosensitivity in Anaplastic Thyroid Carcinoma

Purpose: Anaplastic thyroid carcinoma (ATC) is one of the most invasive human cancers and has a poor prognosis. Molecular targets of ATC that determine its highly aggressive nature remain unidentified. This study investigated L1 cell adhesion molecule (L1CAM) expression and its role in tumorigenesis of ATCs. Experimental Design: Expression of L1CAM in thyroid cancer was evaluated by immunohistochemical analyses of tumor samples from patients with thyroid cancer. We investigated the role of L1CAM in proliferation, migration, invasion, and chemoresistance using short hairpin RNA (shRNA) knockdown experiments in human ATC cell lines. Finally, we evaluated the role of L1CAM on tumorigenesis with ATC xenograft assay in a nude mouse model. Results: L1CAM expression was not detectable in normal follicular epithelial cells of the thyroid or in differentiated thyroid carcinoma. In contrast, analysis of ATC samples showed specifically higher expression of L1CAM in the invasive area of the tumor. Specific knockdown of L1CAM in the ATC cell lines, FRO and 8505C, caused a significant decrease in the proliferative, migratory, and invasive capabilities of the cells. Suppression of L1CAM expression in ATC cell lines increased chemosensitivity to gemcitabine or paclitaxel. Finally, in an ATC xenograft model, depletion of L1CAM markedly reduced tumor growth and increased the survival of tumor-bearing mice. Conclusions: We report that L1CAM is highly expressed in the samples taken from patients with ATCs. L1CAM plays an important role in determining tumor behavior and chemosensitivity in cell lines derived from ATCs. Therefore, we suggest that L1CAM may be an important therapeutic target in patients with ATCs. Clin Cancer Res; 18(11); 3071–8. ©2012 AACR.

Mitochondrial oxidative phosphorylation reserve is required for hormone- and PPARγ agonist-induced adipogenesis

Adipocyte differentiation requires the coordinated activities of several nuclear transcription factors. Recently, mitochondria biogenesis was reported to occur during adipocyte differentiation and following treatment with thiazolidinediones in vitro and in vivo. Crif1 is a translational factor for mitochondrial DNA (mtDNA) and is important for transcription of the mitochondrial oxidative phosphorylation (OXPHOS) complex. To investigate the role of OXPHOS in adipogenesis, we analyzed adipocyte differentiation following disruption of Crif1 in vitro and in vivo. The adipose-specific Crif1 knockout mouse had a lower body weight and less fat mass than wild-type mice. Furthermore, adipocytes were smaller and had a dysplastic morphology in the adipose-specific Crif1 knockout mouse. 3T3-L1 adipocytes or adipose-derived stem cells (ADSCs) that lacked Crif1 expressed lower levels of mtDNA-encoded OXPHOS subunits, and adipocyte differentiation was disrupted. Rosiglitazone treatment did not induce adipogenesis or mitochondria biogenesis in Crif1 knockout ADSCs. These results show that mitochondrial OXPHOS and Crif1 are required for rosiglitazone- and hormone-induced adipogenesis.

Suppression of mitochondrial respiration with auraptene inhibits the progression of renal cell carcinoma: involvement of HIF-1α degradation

Renal cell carcinoma (RCC) progression resulting from the uncontrolled migration and enhanced angiogenesis is an obstacle to effective therapeutic intervention. Tumor metabolism has distinctive feature called Warburg effect, which enhances the aerobic glycolysis rapidly supplying the energy for migration of tumor. To manipulate this metabolic change characteristic of aggressive tumors, we utilized the citrus extract, auraptene, known as a mitochondrial inhibitor, testing its anticancer effects against the RCC4 cell line. We found that auraptene impaired RCC4 cell motility through reduction of mitochondrial respiration and glycolytic pathway-related genes. It also strongly disrupted VEGF-induced angiogenesis in vitro and in vivo. Hypoxia-inducible factor 1a (HIF-1a), a key regulator of cancer metabolism, migration and angiogenesis that is stably expressed in RCCs by virtue of a genetic mutation in the von Hippel–Lindau (VHL) tumor-suppressor protein, was impeded by auraptene, which blocked HIF-1a translation initiation without causing cytotoxicity. We suggest that blockade HIF-1a and reforming energy metabolism with auraptene is an effective approach for suspension RCC progression.