Seung-Whan Kim

Essential Role of Mitochondrial Function in Adiponectin Synthesis in Adipocytes

OBJECTIVE—Adiponectin is an important adipocytokine that improves insulin action and reduces atherosclerotic processes. The plasma adiponectin level is paradoxically reduced in obese individuals, but the underlying mechanism is unknown. This study was undertaken to test the hypothesis that mitochondrial function is linked to adiponectin synthesis in adipocytes. RESEARCH DESIGN AND METHODS—We examined the effects of rosiglitazone and the measures that increase or decrease mitochondrial function on adiponectin synthesis. We also examined the molecular mechanism by which changes in mitochondrial function affect adiponectin synthesis. RESULTS—Adiponectin expression and mitochondrial content in adipose tissue were reduced in obese db/db mice, and these changes were reversed by the administration of rosiglitazone. In cultured adipocytes, induction of increased mitochondrial biogenesis (via adenoviral overexpression of nuclear respiratory factor-1) increased adiponectin synthesis, whereas impairment in mitochondrial function decreased it. Impaired mitochondrial function increased endoplasmic reticulum (ER) stress, and agents causing mitochondrial or ER stress reduced adiponectin transcription via activation of c-Jun NH2-terminal kinase (JNK) and consequent induction of activating transcription factor (ATF)3. Increased mitochondrial biogenesis reversed all of these changes. CONCLUSIONS—Mitochondrial function is linked to adiponectin synthesis in adipocytes, and mitochondrial dysfunction in adipose tissue may explain decreased plasma adiponectin levels in obesity. Impaired mitochondrial function activates a series of mechanisms involving ER stress, JNK, and ATF3 to decrease adiponectin synthesis.

Reversal of P-glycoprotein-mediated multidrug resistance by ginsenoside Rg3

Multidrug resistance has been a major problem in cancer chemotherapy. In this study, in vitro and in vivo modulations of MDR by ginsenoside Rg(3), a red ginseng saponin, were investigated. In flow cytometric analysis using rhodamine 123 as an artificial substrate, Rg(3) promoted accumulation of rhodamine 123 in drug-resistant KBV20C cells in a dose-dependent manner, but it had no effect on parental KB cells. Additionally Rg(3) inhibited [3H]vinblastine efflux and reversed MDR to doxorubicin, COL, VCR, and VP-16 in KBV20C cells. Reverse transcriptase-polymerase chain reaction and immuno-blot analysis after exposure of KBV20C cells to Rg(3) showed that inhibition of drug efflux by Rg(3) was due to neither repression of MDR1 gene expression nor Pgp level. Photo-affinity labeling study with [3H]azidopine, however, revealed that Rg(3) competed with [3H]azidopine for binding to the Pgp demonstrating that Rg(3) competed with anticancer drug for binding to Pgp thereby blocking drug efflux. Furthermore, Rg(3) increased life span in mice implanted with DOX-resistant murine leukemia P388 cells in vivo and inhibited body weight increase significantly.

Effect of Heat Shock and Mutations in ClpL and ClpP on Virulence Gene Expression in Streptococcus pneumoniae

ABSTRACT Spread of Streptococcus pneumoniae from the nasopharynx to other host tissues would require the organism to adapt to a variety of environmental conditions. Since heat shock proteins are induced by environmental stresses, we investigated the effect of heat shock on ClpL and ClpP synthesis and the effect of clpL and clpP mutations on the expression of key pneumococcal virulence genes. Pulse labeling with [35S]methionine and chase experiments as well as immunoblot analysis demonstrated that ClpL, DnaK, and GroEL were stable. Purified recombinant ClpL refolded urea-denatured rhodanese in a dose-dependent manner, demonstrating ClpLs chaperone activity. Although growth of the clpL mutant was not affected at 30 or 37°C, growth of the clpP mutant was severely affected at these temperatures. However, both clpL and clpP mutants were sensitive to 43°C. Although it was further induced by heat shock, the level of expression of ClpL in the clpP mutant was high at 30°C, suggesting that ClpP represses expression of ClpL. Furthermore, the clpP mutation significantly attenuated the virulence of S. pneumoniae in a murine intraperitoneal infection model, whereas the clpL mutation did not. Interestingly, immunoblot and real-time reverse transcription-PCR analysis demonstrated that pneumolysin and pneumococcal surface antigen A were induced by heat shock in wild-type S. pneumoniae. Other virulence genes were also affected by heat shock and clpL and clpP mutations. Virulence gene expression seems to be modulated not only by heat shock but also by the ClpL and ClpP proteases.

Autophagy deficiency in beta cells leads to compromised unfolded protein response and progression from obesity to diabetes in mice

Aims/hypothesisThe unfolded protein response (UPR) in endoplasmic reticulum (ER) and autophagy are known to be related. We investigated the role of autophagy in UPR of pancreatic beta cells and the susceptibility of autophagy-deficient beta cells to the ER stress that is implicated in the development of diabetes.MethodsRat insulin promoter (RIP)-Cre+;autophagy-related 7 (Atg7)F/W mice were bred with ob/w mice to derive RIP-Cre+;Atg7F/F-ob/ob mice and to induce ER stress in vivo. GFP-LC3+-ob/ob mice were generated to examine in vivo autophagic activity. Real-time RT-PCR was performed to study the expression of the genes of the UPR machinery. Proteolysis was assessed by determining release of incorporated radioactive leucine.ResultsProduction of UPR machinery was reduced in autophagy-deficient beta cells, which was associated with diminished production of p85α and p85β regulatory subunits of phosphoinositide 3-kinase. Because of compromised UPR machinery, autophagy-deficient beta cells were susceptible to ER stressors in vitro. When mice with beta cell-specific autophagy deficiency, which have mild hyperglycaemia, were bred with ob/ob mice to induce ER stress in vivo, severe diabetes developed, which was accompanied by an increase in beta cell death and accumulation of reactive oxygen species. The increased demand for UPR present in obesity was unmet in autophagy-deficient beta cells. Autophagy level and autophagic activity were enhanced by lipid, while proteolysis was reduced.Conclusions/interpretationThese results suggest that autophagy is important for intact UPR machinery and appropriate UPR in response to lipid injury that increases demand for UPR. Autophagy deficiency in pancreatic beta cells may contribute to the progression from obesity to diabetes.

AMP kinase acts as a negative regulator of RANKL in the differentiation of osteoclasts.

INTRODUCTION AMP-activated protein kinase (AMPK) has been reported to stimulate differentiation and proliferation of osteoblasts, but the role of AMPK in the physiology of osteoclasts has not been investigated. METHOD Osteoclasts were differentiated from mouse BMMϕs. TRAP-positive multinucleated cells were considered to be osteoclasts using TRAP staining, and resorption area was determined by incubation of cells on dentine discs. Signaling pathways were investigated using Western blotting and RT-PCR. RESULTS RANKL induced phosphorylation/activation of AMPK-α in BMMϕs and stimulated formation of TRAP-positive multinucleated cells. Pharmacological inhibition of AMPK with compound C and siRNA-mediated knockdown of AMPK-α1, the predominant α-subunit isoform in BMMϕs, increased RANKL-induced formation of TRAP-positive multinucleated cells and bone resorption via activation of the downstream signaling elements p38, JNK, NF-κB, Akt, CREB, c-Fos, and NFATc1. STO-609, an inhibitor of CaMKK, completely blocked the RANKL-induced activation of AMPK-α, but KN-93, an inhibitor of CaMK, did not. siRNA-mediated TAK1 knockdown also blocked RANKL-induced activation of AMPK-α. The AMPK activators metformin, (-)-epigallocatechin-3-gallate, berberine, resveratrol, and α-lipoic acid dose-dependently suppressed formation of TRAP-positive multinucleated cells and bone resorption. CONCLUSION AMPK negatively regulates RANKL, possibly by acting through CaMKK and TAK1. Thus, the development of AMPK activators may be a useful strategy for inhibiting the resorption of bone that is stimulated under RANKL-activated conditions.

NF-κB activation in hypothalamic POMC neurons is essential in illness- and leptin-induced anorexia

Anorexia and weight loss are prevalent in infectious diseases. To investigate the molecular mechanisms underlying these phenomena, we established animal models of infection-associated anorexia by administrating bacterial and viral products, lipopolysaccharide (LPS) and human immunodeficiency virus-1 transactivator protein (Tat). In these models, we found that the nuclear factor-κB (NF-κB), a pivotal transcription factor for inflammation-related proteins, was activated in the hypothalamus. In parallel, administration of LPS and Tat increased hypothalamic pro-inflammatory cytokine production, which was abrogated by inhibition of hypothalamic NF-κB. In vitro, NF-κB activation directly stimulated the transcriptional activity of pro-opiomelanocortin (POMC), a precursor of anorexigenic melanocortin, and mediated the stimulatory effects of LPS, Tat, and pro-inflammatory cytokines on POMC transcription, implying the involvement of NF-κB in controlling feeding behavior. Consistently, hypothalamic injection of LPS and Tat caused a significant reduction in food intake and body weight, which was prevented by blockade of NF-κB and melanocortin. Furthermore, disruption of IκB kinase-β, an upstream kinase of NF-κB, in POMC neurons attenuated LPS- and Tat-induced anorexia. These findings suggest that infection-associated anorexia and weight loss are mediated via NF-κB activation in hypothalamic POMC neurons. In addition, hypothalamic NF-κB was activated by leptin, an important anorexigenic hormone, and mediates leptin-stimulated POMC transcription, indicating that hypothalamic NF-κB also serves as a downstream signaling pathway of leptin.

Limited stress response in Streptococcus pneumoniae

In Streptococcus pneumoniae, heat shock induces the synthesis of 65‐, 73‐, and 84‐kDa proteins, and ethanol shock induces a 104‐kDa protein. In this study, the 65‐, 84‐, and 104‐kDa proteins were identified as members of the GroEL, ClpL and alcohol dehydrogenase families, respectively, and the general properties of the stress response of S. pneumoniae to several other stresses were characterized. However, several stresses which are known to induce stress responses in Escherichia coli and Bacillus subtilis failed to induce any high molecular weight heat‐shock proteins (HSPs) such as GroEL and DnaK homologues. A minor temperature shift from 30 to 37 C triggered induction of the homologues of DnaK and GroEL of E. coli. These features may provide a foundation for evaluating the role of heat‐shock proteins relative to the physiology and pathogenesis of pneumococcus.

Characterization of ASC-2 as an antiatherogenic transcriptional coactivator of liver X receptors in macrophages.

Activating signal cointegrator-2 (ASC-2) functions as a transcriptional coactivator of many nuclear receptors and also plays important roles in the physiology of the liver and pancreas by interacting with liver X receptors (LXRs), which antagonize the development of atherosclerosis. This study was undertaken to establish the specific function of ASC-2 in macrophages and atherogenesis. Intriguingly, ASC-2 was more highly expressed in macrophages than in the liver and pancreas. To inhibit LXR-specific activity of ASC-2, we used DN2, which contains the C-terminal LXXLL motif of ASC-2 and thereby acts as an LXR-specific, dominant-negative mutant of ASC-2. In DN2-overexpressing transgenic macrophages, cellular cholesterol content was higher and cholesterol efflux lower than in control macrophages. DN2 reduced LXR ligand-dependent increases in the levels of ABCA1, ABCG1, and apolipoprotein E (apoE) transcripts as well as the activity of luciferase reporters driven by the LXR response elements (LXREs) of ABCA1, ABCG1, and apoE genes. These inhibitory effects of DN2 were reversed by overexpression of ASC-2. Chromatin immunoprecipitation analysis demonstrated that ASC-2 was recruited to the LXREs of the ABCA1, ABCG1, and apoE genes in a ligand-dependent manner and that DN2 interfered with the recruitment of ASC-2 to these LXREs. Furthermore, low-density lipoprotein receptor (LDLR)-null mice receiving bone marrow transplantation from DN2-transgenic mice showed accelerated atherogenesis when administered a high-fat diet. Taken together, these results indicate that suppression of the LXR-specific activity of ASC-2 results in both defective cholesterol metabolism in macrophages and accelerated atherogenesis, suggesting that ASC-2 is an antiatherogenic coactivator of LXRs in macrophages.

Hepatic TRAP80 selectively regulates lipogenic activity of liver X receptor

Inflammation in response to excess low-density lipoproteins in the blood is an important driver of atherosclerosis development. Due to its ability to enhance ATP-binding cassette A1-dependent (ABCA1-dependent) reverse cholesterol transport (RCT), liver X receptor (LXR) is an attractive target for the treatment of atherosclerosis. However, LXR also upregulates the expression of sterol regulatory element-binding protein 1c (SREBP-1c), leading to increased hepatic triglyceride synthesis, an independent risk factor for atherosclerosis. Here, we developed a strategy to separate the favorable and unfavorable effects of LXR by exploiting the specificity of the coactivator thyroid hormone receptor-associated protein 80 (TRAP80). Using human hepatic cell lines, we determined that TRAP80 selectively promotes the transcription of SREBP-1c but not ABCA1. Adenovirus-mediated expression of shTRAP80 inhibited LXR-dependent SREBP-1c expression and RNA polymerase II recruitment to the LXR responsive element (LXRE) of SREBP-1c, but not to the LXRE of ABCA1. In murine models, liver-specific knockdown of TRAP80 ameliorated liver steatosis and hypertriglyceridemia induced by LXR activation and maintained RCT stimulation by the LXR ligand. Together, these data indicate that TRAP80 is a selective regulator of hepatic lipogenesis and is required for LXR-dependent SREBP-1c activation. Moreover, targeting the interaction between TRAP80 and LXR should facilitate the development of potential LXR agonists that effectively prevent atherosclerosis.

SREBP-1c regulates glucose-stimulated hepatic clusterin expression.

Clusterin is a stress-response protein that is involved in diverse biological processes, including cell proliferation, apoptosis, tissue differentiation, inflammation, and lipid transport. Its expression is upregulated in a broad spectrum of diverse pathological states. Clusterin was recently reported to be associated with diabetes, metabolic syndrome, and their sequelae. However, the regulation of clusterin expression by metabolic signals was not addressed. In this study we evaluated the effects of glucose on hepatic clusterin expression. Interestingly, high glucose concentrations significantly increased clusterin expression in primary hepatocytes and hepatoma cell lines, but the conventional promoter region of the clusterin gene did not respond to glucose stimulation. In contrast, the first intronic region was transcriptionally activated by high glucose concentrations. We then defined a glucose response element (GlRE) of the clusterin gene, showing that it consists of two E-box motifs separated by five nucleotides and resembles carbohydrate response element (ChoRE). Unexpectedly, however, these E-box motifs were not activated by ChoRE binding protein (ChREBP), but were activated by sterol regulatory element binding protein-1c (SREBP-1c). Furthermore, we found that glucose induced recruitment of SREBP-1c to the E-box of the clusterin gene intronic region. Taken together, these results suggest that clusterin expression is increased by glucose stimulation, and SREBP-1c plays a crucial role in the metabolic regulation of clusterin.