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Mitochondrial dysfunction is considered a pathogenic linker in the development of non-alcoholic steatohepatitis (NASH). Inappropriate mitochondrial protein-quality control, possibly induced by insufficiency of the mitochondrial matrix caseinolytic protease P (ClpP), can potentially cause mitochondrial dysfunction. Herein, we aimed to investigate hepatic ClpP levels in a diet-induced model of NASH and determine whether supplementation of ClpP can ameliorate diet-induced NASH.NASH was induced by a high-fat/high-fructose (HF/HFr) diet in C57BL/6J mice. Stress/inflammatory signals were induced in mouse primary hepatocytes (MPHs) by treatment with palmitate/oleate (PA/OA). ClpP levels in hepatocytes were reduced using the RNAi-mediated gene knockdown technique but increased through the viral transduction of ClpP. ClpP activation was induced by administering a chemical activator of ClpP.Hepatic ClpP protein levels in C57BL/6J mice fed a HF/HFr diet were lower than the levels in those fed a normal chow diet. PA/OA treatment also decreased the ClpP protein levels in MPHs. Overexpression or activation of ClpP reversed PA/OA-induced mitochondrial dysfunction and stress/inflammatory signal activation in MPHs, whereas ClpP knockdown induced mitochondrial dysfunction and stress/inflammatory signals in these cells. On the other hand, ClpP overexpression or activation improved HF/HFr-induced NASH characteristics such as hepatic steatosis, inflammation, fibrosis, and injury in the C57BL/6J mice, whereas ClpP knockdown further augmented steatohepatitis in mice fed a HF/HFr diet.Reduced ClpP expression and subsequent mitochondrial dysfunction are key to the development of diet-induced NASH. ClpP supplementation through viral transduction or chemical activation represents a potential therapeutic strategy to prevent diet-induced NASH.Western diets, containing high fat and high fructose, often induce non-alcoholic steatohepatitis (NASH). Mitochondrial dysfunction is considered pathogenically linked to diet-induced NASH. We observed that the mitochondrial protease ClpP decreased in the livers of mice fed a western diet and supplementation of ClpP ameliorated western diet-induced NASH.

Non-alcoholic fatty liver disease (NAFLD) includes a broad spectrum of liver diseases characterized by steatosis, inflammation, and fibrosis. This study aimed to investigate the potential of dipeptidyl peptidase-4 inhibitors and sodium-glucose cotransporter 2 inhibitors in alleviating the progression of NAFLD. The NAFLD model was generated by feeding male C57BL/6J mice a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) for 7 weeks. After 2 weeks of CDAHFD feeding, the NAFLD model mice were assigned to four groups, namely (ⅰ) VEHICLE, (ⅱ) gemigliptin (GEMI), (ⅲ) empagliflozin (EMPA), and (ⅳ) GEMI + EMPA. For the next 5 weeks, mice received the vehicle or the drug based upon the group to which they belonged. Thereafter, the triglyceride concentration, extent of fibrosis, and the expression of genes encoding inflammatory cytokines, chemokines, and antioxidant enzymes were analyzed in the livers of mice. The NAFLD activity score and hepatic fibrosis grade were assessed via hematoxylin and eosin and Sirius Red staining of the liver tissue samples. All mice belonging to the GEMI, EMPA, and GEMI + EMPA groups showed improvements in the accumulation of liver triglycerides and the expression of inflammatory cytokines and chemokines. Additionally, the oxidative stress was reduced due to inhibition of the c-Jun N-terminal kinase pathway and upregulation of the antioxidant enzymes. Furthermore, in these three groups, the galectin-3 and interleukin 33-induced activity of tumor necrosis factor-α was inhibited, thereby preventing the progression of liver fibrosis. These findings suggest that the GEMI, EMPA, and GEMI + EMPA treatments ameliorate hepatic steatosis, inflammation, oxidative stress, and fibrosis in CDAHFD-induced NAFLD mouse models.

Background Nanoparticles have been utilized in brain research and therapeutics, including imaging, diagnosis, and drug delivery, owing to their versatile properties compared to bulk materials. However, exposure to nanoparticles leads to their accumulation in the brain, but drug development to counteract this nanotoxicity remains challenging. To date, concerns have risen about the potential toxicity to the brain associated with nanoparticles exposure via penetration of the brain blood barrier to address this issue. Methods Here the effect of silica-coated-magnetic nanoparticles containing the rhodamine B isothiocyanate dye [MNPs@SiO2(RITC)] were assessed on microglia through toxicological investigation, including biological analysis and integration of transcriptomics, proteomics, and metabolomics. MNPs@SiO2(RITC)-induced biological changes, such as morphology, generation of reactive oxygen species, intracellular accumulation of MNPs@SiO2(RITC) using transmission electron microscopy, and glucose uptake efficiency, were analyzed in BV2 murine microglial cells. Each omics data was collected via RNA-sequencing-based transcriptome analysis, liquid chromatography-tandem mass spectrometry-based proteome analysis, and gas chromatography- tandem mass spectrometry-based metabolome analysis. The three omics datasets were integrated and generated as a single network using a machine learning algorithm. Nineteen compounds were screened and predicted their effects on nanotoxicity within the triple-omics network. Results Intracellular reactive oxygen species production, an inflammatory response, and morphological activation of cells were greater, but glucose uptake was lower in MNPs@SiO2(RITC)-treated BV2 microglia and primary rat microglia in a dose-dependent manner. Expression of 121 genes (from 41,214 identified genes), and levels of 45 proteins (from 5918 identified proteins) and 17 metabolites (from 47 identified metabolites) related to the above phenomena changed in MNPs@SiO2(RITC)-treated microglia. A combination of glutathione and citrate attenuated nanotoxicity induced by MNPs@SiO2(RITC) and ten other nanoparticles in vitro and in the murine brain, protecting mostly the hippocampus and thalamus. Conclusions Combination of glutathione and citrate can be one of the candidates for nanotoxicity alleviating drug against MNPs@SiO2(RITC) induced detrimental effect, including elevation of intracellular reactive oxygen species level, activation of microglia, and reduction in glucose uptake efficiency. In addition, our findings indicate that an integrated triple omics approach provides useful and sensitive toxicological assessment for nanoparticles and screening of drug for nanotoxicity. Graphical Abstract

The accumulation of hepatic lipid droplets (LDs) is a hallmark of non-alcoholic fatty liver disease (NAFLD). Appropriate degradation of hepatic LDs and oxidation of complete free fatty acids (FFAs) are important for preventing the development of NAFLD. Histone deacetylase (HDAC) is involved in the impaired lipid metabolism seen in high-fat diet (HFD)-induced obese mice. Here, we evaluated the effect of MS-275, an inhibitor of HDAC1/3, on the degradation of hepatic LDs and FFA oxidation in HFD-induced NAFLD mice. To assess the dynamic degradation of hepatic LDs and FFA oxidation in fatty livers of MS-275-treated HFD C57BL/6J mice, an intravital two-photon imaging system was used and biochemical analysis was performed. The MS-275 improved hepatic metabolic alterations in HFD-induced fatty liver by increasing the dynamic degradation of hepatic LDs and the interaction between LDs and lysozyme in the fatty liver. Numerous peri-droplet mitochondria, lipolysis, and lipophagy were observed in the MS-275-treated mouse fatty liver. Biochemical analysis revealed that the lipolysis and autophagy pathways were activated in MS-275 treated mouse liver. In addition, MS-275 reduced the de novo lipogenesis, but increased the mitochondrial oxidation and the expression levels of oxidation-related genes, such as PPARa, MCAD, CPT1b, and FGF21. Taken together, these results suggest that MS-275 stimulates the degradation of hepatic LDs and mitochondrial free fatty acid oxidation, thus protecting against HFD-induced NAFLD.

Background. Sodium-glucose cotransporter 2 (SGLT2) and dipeptidyl peptidase-4 (DPP-4) inhibitors are glucose-lowering drugs whose anti-inflammatory properties have recently become useful in tackling metabolic syndromes in chronic inflammatory diseases, including diabetes and obesity. We investigated whether empagliflozin (SGLT2 inhibitor) and gemigliptin (DPP-4 inhibitor) improve inflammatory responses in macrophages, identified signalling pathways responsible for these effects, and studied whether the effects can be augmented with dual empagliflozin and gemigliptin therapy. Methods. RAW 264.7 macrophages were first stimulated with lipopolysaccharide (LPS), then cotreated with empagliflozin, gemigliptin, or empagliflozin plus gemigliptin. We conducted quantitative RT-PCR (qRT-PCR) to determine the most effective anti-inflammatory doses without cytotoxicity. We performed ELISA and qRT-PCR for inflammatory cytokines and chemokines and flow cytometry for CD80, the M1 macrophage surface marker, to evaluate the anti-inflammatory effects of empagliflozin and gemigliptin. NF-κB, MAPK, and JAK2/STAT signalling pathways were examined via Western blotting to elucidate the molecular mechanisms of anti-inflammation. Results. LPS-stimulated CD80+ M1 macrophages were suppressed by coincubation with empagliflozin, gemigliptin, and empagliflozin plus gemigliptin, respectively. Empagliflozin and gemigliptin (individually and combined) inhibited prostaglandin E2 (PGE2) release and COX-2, iNOS gene expression in LPS-stimulated RAW 264.7 macrophages. These three treatments also attenuated the secretion and mRNA expression of proinflammatory cytokines, such as TNF-α, IL-1β, IL-6, and IFN-γ, and proinflammatory chemokines, such as CCL3, CCL4, CCL5, and CXCL10. All of them blocked NF-κB, JNK, and STAT1/3 phosphorylation through IKKα/β, MKK4/7, and JAK2 signalling. Conclusions. Our study demonstrated the anti-inflammatory effects of empagliflozin and gemigliptin via IKK/NF-κB, MKK7/JNK, and JAK2/STAT1 pathway downregulation in macrophages. In all cases, combined empagliflozin and gemigliptin treatment showed greater anti-inflammatory properties.

Background and aim Non-alcoholic fatty liver disease (NAFLD) ranges from simple steatosis to non-alcoholic steatohepatitis, which is characterized by hepatic inflammation that can progress to fibrosis, cirrhosis, and hepatocellular carcinoma. Visfatin, an adipocytokine, was reported to induce pro-inflammatory cytokines and can be associated with liver fibrosis. We investigated the role of visfatin on hepatic inflammation and fibrosis in a methionine-choline-deficient (MCD)-diet-induced steatohepatitis mouse model. Methods Eight-week-old male C57BL/6 J mice were randomly assigned into one of three groups: (1) saline-injected control diet group; (2) saline-injected MCD diet group; and (3) visfatin-injected MCD diet group (n = 8 per group). Mice were administered intravenous saline or 10 μg/kg of recombinant murine visfatin for 2 weeks. Histologic assessment of liver and biochemical and molecular measurements of endoplasmic reticulum (ER) stress, reactive oxidative stress (ROS), inflammation, and fibrosis were performed in livers from these animals. Results Visfatin injection aggravated hepatic steatosis and increased plasma alanine aminotransferase and aspartate aminotransferase concentrations. Visfatin increased inflammatory cell infiltration (as indicated by F4/80, CD68, ly6G, and CD3 mRNA expression) and expression of chemokines in the liver. Visfatin also increased the expression of pro-inflammatory cytokines (IL-1β, TNF-α, and IL-6) and activated fibrosis markers (CTGF, TIMP1, collagen 1α2, collagen 3α2, αSMA, fibronectin, and vimentin) in liver. Livers of visfatin-injected mice showed upregulation of ER stress and ROS and activation of JNK signaling. Conclusions These results suggest that visfatin aggravates hepatic inflammation together with induction of ER and oxidative stress and exacerbates fibrosis in an MCD-diet-fed mouse model of NAFLD.

Chemokines interact with hepatic resident cells during inflammation and fibrosis. CC chemokine ligand (CCL) 20 has been reported to be important in inflammation and fibrosis in the liver. We hypothesized that visfatin, an adipocytokine, could play a role in hepatic fibrosis via CCL20. We investigated the effect of visfatin on CCL20 in THP-1 human promonocytic cells and examined the molecular mechanisms involved. Following treatment of THP-1 cells with visfatin, CCL20 expression and secretion were assessed. We assessed the intracellular signaling molecules IKK/NF-κB, JAK2/STAT3, MAPKs, and MKK3/6 by western blotting. We treated THP-1 cells with visfatin and signaling inhibitors, and examined CCL20 mRNA and protein levels. To investigate the effect of visfatin-induced CCL20 expression in hepatic stellate cells (HSCs), LX-2 cells were co-cultured with the culture supernatant of THP-1 cells with or without anti-CCL20 neutralizing antibodies, and fibrosis markers were examined by RT-PCR and immunoblotting. In THP-1 cells, visfatin increased the CCL20 mRNA and protein levels. visfatin increased the activities of the NF-κB, p38, and MLK3/6 signaling pathways but not those of the JAK2/STAT3 and ERK pathways. Visfatin treatment together with an NF-κB, p38, or MLK3 inhibitor reduced the mRNA and protein levels of CCL20. The visfatin-induced CCL20 increased the expression of fibrosis markers and CCR6 in HSCs. Following neutralization of CCL20, the levels of fibrosis markers and CCR6 were decreased. Visfatin increases the expression of CCL20 via the NF-κB and MKK3/6-p38 signaling pathways in macrophages, and visfatin-induced CCL20 expression promotes the fibrosis markers in HSCs.

It is known that hyperlipidemia is a risk factor for sensorineural hearing loss. However, the biological mechanisms underlying hyperlipidemia and hearing impairment have not been completely elucidated in the cochlea. Based on our previous study of human subjects, elderly people taking drugs for hyperlipidemia showed better hearing than those not taking any medications. We hypothesized that drugs for hyperlipidemia, such as statins, may have the potential to prevent hearing impairment. The aim of this study was to investigate the correlation between hyperlipidemia and hearing impairment and the hearing preservation effect of atorvastatin using a hyperlipidemic mouse model with diet-induced obesity (DIO). Here, we demonstrate that DIO mice had a significant hearing impairment as well as increased levels of reactive oxygen species (ROS) and hair cell death due to reduced levels of pAKT and superoxide dismutase 2 (SOD2). However, these changes were significantly prevented by atorvastatin. In mechanistic studies, oxidative stress-induced intrinsic apoptosis was decreased by the high expression of nrf2 and antioxidant genes, which improved mitochondrial function and ROS via activation of the PI3K-AKT pathway by atorvastatin. Therefore, atorvastatin has the potential to prevent hearing impairment via redox balance in the presence of hyperlipidemia.

Background. It has been suggested that visfatin, which is an adipocytokine, exhibits proinflammatory properties and is associated with insulin resistance. Insulin resistance and inflammation are the principal pathogeneses of nonalcoholic fatty liver disease (NAFLD), but the relationship, if any, between visfatin and NAFLD remains unclear. Here, we evaluated the effects of visfatin on hepatic inflammation and insulin resistance in HepG2 cells and examined the molecular mechanisms involved. Methods. After treatment with visfatin, the inflammatory cytokines IL-6, TNF-α, and IL-1β were assessed by real-time polymerase chain reaction (RT-PCR) and immunocytochemical staining in HepG2 cells. To investigate the effects of visfatin on insulin resistance, we evaluated insulin-signaling pathways, such as IR, IRS-1, GSK, and AKT using immunoblotting. We assessed the intracellular signaling molecules including STAT3, NF-κB, IKK, p38, JNK, and ERK by western blotting. We treated HepG2 cells with both visfatin and either AG490 (a JAK2 inhibitor) or Bay 7082 (an NF-κB inhibitor); we examined proinflammatory cytokine mRNA levels using RT-PCR and insulin signaling using western blotting. Results. In HepG2 cells, visfatin significantly increased the levels of proinflammatory cytokines, reduced the levels of proteins (e.g., phospho-IR, phospho-IRS-1 (Tyr612), phospho-AKT, and phospho-GSK-3α/β) involved in insulin signaling, and increased IRS-1 S307 phosphorylation compared to controls. Interestingly, visfatin increased the activities of the JAK2/STAT3 and IKK/NF-κB signaling pathways but not those of the JNK, p38, and ERK pathways. Visfatin-induced inflammation and insulin resistance were regulated by JAK2/STAT3 and IKK/NF-κB signaling; together with AG490 or Bay 7082, visfatin significantly reduced mRNA levels of IL-6, TNF-α and IL-1β and rescued insulin signaling. Conclusion. Visfatin induced proinflammatory cytokine production and inhibited insulin signaling via the STAT3 and NF-κB pathways in HepG2 cells.

Aim: This study was designed to evaluate the effects of SGLT2 inhibitor on palmitate-induced lipotoxicity of cardiomyocytes and the underlying mechanism involved with palmitate. Methods: We checked SGLT2 expression in muscle cells, including H9C2. H9C2 cells were palmitate treated with/without SGLT2 inhibitor. Then, insulin resistance was measured, 2-NBDG uptake, and immunoblotting using insulin signaling pathway antibodies. Inflammation and cardiac metabolism were analyzed several related genes. Cardiomyocyte apoptosis was also detected using DNA fragmentations assay and immunoblotting using cleaved caspase 3. Results: Low doses of palmitates evoked lipotoxicity in cardiomyocytes. Treatment of SGLT2 inhibitor dramatically reduced palmitate-induced expression levels of inflammatory cytokines. Immunoblotting analysis showed treatment of cardiomyocytes, with SGLT2 inhibitor, recovered impaired insulin signaling, such as phospho-IR and phospho-AKT, but total IR and AKT did not change. SGLT2 inhibitor also restored insulin-stimulated glucose uptake, which was destroyed by lipotoxicity. Treatment with SGLT2 inhibitor decreased the level of ER stress markers as well as inflammatory signaling markers. In particular, SGLT2 inhibitor stimulated phosphorylation of AMPK and fatty acid beta-oxidation. Cardiomyocyte exposure to high doses of palmitates evoked apoptotic features such as caspase 3 activation and DNA fragmentations. But pretreatment of SGLT2 inhibitor significantly protected palmitate-induced apoptotic features. Conclusions: This data suggests SGLT2 inhibitors protect palmitate-induced cardiomyocytes inflammation, impaired cardiac metabolism, insulin resistance, and cardiomyocyte apoptosis. Therefore, attempts to use treatment of SGLT2 inhibitor might be a useful strategy for preventing diabetes-associated ventricular remodeling and diabetic cardiac complication. Disclosure H. Lee: None. S. Choi: None. Y. Kang: None. T. Kim: None. N. Lee: None. H. Moon: None. S. Han: None. D. Kim: None. K. Lee: None.

Aim/Hypotheses: Iron plays an important role in many physiological processes, including redox balance, inflammation, and mitochondrial respiratory metabolism. Abnormalities in iron metabolism lead to insulin resistance as well as diabetes. However, the abnormal mechanism of iron metabolism in the muscles of insulin resistance animal models is not precisely well known. Therefore, we investigated the molecular mechanism underlying iron dysregulation in high fat diet-induced C57BL/6J mice skeletal muscles. Methods: We measured intracellular iron content in skeletal muscle tissues and iron metabolism related protein levels, such as transferrin receptor1, heavy chain ferritin, light chain ferritin, DMT1, and ferroportin. To investigate the association between iron content and insulin resistance, we treated iron donor or chelator in an animal model and performed insulin tolerance tests and measurement of insulin signaling. Results: Interestingly, intracellular iron levels were increased in high fat diet-induced mice skeletal muscles. We also observed up-regulation of transferrin receptor 1 (TfR1), ferritin, but did not observe a change of DMT1 and ferroportin. Treatment with iron dextran, an iron donor, in C57BL/6J normal mice induced insulin resistance. However, treatment of desferrioxamine, an iron chelator, in high fat diet-induced insulin resistance mice recovered insulin resistance. Iron overload with iron dextran impaired insulin signaling in skeletal muscles, while iron chelation with desferrioxamine recovered high fat-induced dysregulated insulin signaling. Conclusions/interpretation: Therefore, we consider fine-tuning of skeletal muscle iron levels and highly suggest the use for preventing and/or treating insulin resistance and diabetes. Disclosure S. Choi: None. N. Lee: None. H. Moon: None. H. Kim: None. J. Jeon: None. Y. Kang: None. T. Kim: None. K. Lee: None.

The present study investigated whether glucagon like peptide‑1 (GLP‑1) improves glucose uptake through glucose transporter type 4 (GLUT4), mediated by the activation of sirtuin 1 (SIRT1), in skeletal muscle cells with palmitate induced‑insulin resistance. The levels of glucose uptake, GLUT4, protein kinase A (PKA), and cyclic adenosine monophosphate (cAMP) were determined in human skeletal muscle myotubes (HSMMs) exposed to palmitate and GLP‑1. Then, to determine whether PKA/cAMP were downstream signals of GLP‑1, a PKA inhibitor was used. To determine whether SIRT‑1 contributes to GLP‑1 action in HSMMs with palmitate‑induced insulin resistance, the levels of peroxisome proliferator‑activated receptor γ coactivator 1α (PGC1α) deacetylation and SIRT‑1 activity were assessed using a SIRT1 inhibitor and small interfering RNA (siRNA). The phosphorylation levels of protein kinase B (Akt) and insulin receptor substrate 1 (IRS‑1) as insulin signaling pathways, were assessed in GLP‑1‑treated HSMMs exposed to palmitate. The influence of SIRT1 on the GLP‑1‑induced activation of insulin signaling pathway was determined using a SIRT1 inhibitor. GLP‑1 restored the palmitate‑induced reductions in the levels of glucose uptake, GLUT4 mRNA, GLUT4 promoter activity, and GLUT4 protein in HSMMs. PKA and cAMP, as GLP‑1 downstream signals, played a role in this process. GLP‑1 increased the deacetylation levels of PGC1α, and stimulated SIRT1 in HSMMs. Moreover, the SIRT1 inhibitor and siRNA of SIRT1 suppressed the effect of GLP‑1 on GLUT4 expression in HSMMs exposed to palmitate. The SIRT1 inhibitor also prevented the GLP‑1‑induced phosphorylation of IRS‑1 and Akt in palmitate‑treated HSMMs. The present findings suggest that in palmitate‑induced insulin‑resistant HSMM, GLP‑1 activates SIRT1 through the PKA/cAMP pathway, which in turn enhances glucose uptake through GLUT4 and the insulin signaling pathway.

Free fatty acid is considered to be one of the major pathogenic factors of inducing insulin resistance. The association between iron disturbances and insulin resistance has recently begun to receive a lot of attention. Although skeletal muscles are a major tissue for iron utilization and storage, the role of iron in palmitate (PA)-induced insulin resistance is unknown. We investigated the molecular mechanism underlying iron dysregulation in PA-induced insulin resistance. Interestingly, we found that PA simultaneously increased intracellular iron and induced insulin resistance. The iron chelator deferoxamine dramatically inhibited PA-induced insulin resistance, and iron donors impaired insulin sensitivity by activating JNK. PA up-regulated transferrin receptor 1 (tfR1), an iron uptake protein, which was modulated by iron-responsive element-binding proteins 2. Knockdown of tfR1 and iron-responsive element-binding proteins 2 prevented PA-induced iron uptake and insulin resistance. PA also translocated the tfR1 by stimulating calcium influx, but the calcium chelator, BAPTA-AM, dramatically reduced iron overload by inhibiting tfR1 translocation and ultimately increased insulin sensitivity. Iron overload may play a critical role in PA-induced insulin resistance. Blocking iron overload may thus be a useful strategy for preventing insulin resistance and diabetes.-Cui, R., Choi, S.-E., Kim, T. H., Lee, H. J., Lee, S. J., Kang, Y., Jeon, J. Y., Kim, H. J., Lee, K.-W. Iron overload by transferrin receptor protein 1 regulation plays an important role in palmitate-induced insulin resistance in human skeletal muscle cells.

The circadian clock organizes the physiology and behavior of organisms to their daily environmental rhythms. The central circadian timekeeping mechanism in eukaryotic cells is the transcriptional-translational feedback loop (TTFL). In the Drosophila TTFL, the transcription factors CLOCK (CLK) and CYCLE (CYC) play crucial roles in activating expression of core clock genes and clock-controlled genes. Many signaling pathways converge on the CLK/CYC complex and regulate its activity to fine-tune the cellular oscillator to environmental time cues. We aimed to identify factors that regulate CLK by performing tandem affinity purification combined with mass spectrometry using Drosophila S2 cells that stably express HA/FLAG-tagged CLK and V5-tagged CYC. We identified SNF4Aγ, a homolog of mammalian AMP-activated protein kinase γ (AMPKγ), as a factor that copurified with HA/FLAG-tagged CLK. The AMPK holoenzyme composed of a catalytic subunit AMPKα and two regulatory subunits, AMPKβ and AMPKγ, directly phosphorylated purified CLK in vitro. Locomotor behavior analysis in Drosophila revealed that knockdown of each AMPK subunit in pacemaker neurons induced arrhythmicity and long periods. Knockdown of AMPKβ reduced CLK levels in pacemaker neurons, and thereby reduced pre-mRNA and protein levels of CLK downstream core clock genes, such as period and vrille. Finally, overexpression of CLK reversed the long-period phenotype that resulted from AMPKβ knockdown. Thus, we conclude that AMPK, a central regulator of cellular energy metabolism, regulates the Drosophila circadian clock by stabilizing CLK and activating CLK/CYC-dependent transcription. SIGNIFICANCE STATEMENT Regulation of the circadian transcription factors CLK and CYC is fundamental to synchronize the core clock with environmental changes. Here, we show that the AMPKγ subunit of AMPK, a central regulator of cellular metabolism, copurifies with the CLK/CYC complex in Drosophila S2 cells. Furthermore, the AMPK holoenzyme directly phosphorylates CLK in vitro. This study demonstrates that AMPK activity regulates the core clock in Drosophila by activating CLK, which enhances circadian transcription. In mammals, AMPK affects the core clock by downregulating circadian repressor proteins. It is intriguing to note that AMPK activity is required for core clock regulation through circadian transcription enhancement, whereas the target of AMPK action is different in Drosophila and mammals (positive vs negative element, respectively).

Aim: In this study, we investigated the relationship and molecular mechanisms between iron-overload and ER-stress-induced insulin resistance in human skeletal muscle cells. Methods: Intracellular iron was measured using calcein AM. Iron metabolism-related genes were analyzed by immunoblotting and PCR. The study was conducted using iron supplementation, which was achieved by palmitate, FeSO4 and FeCl3 administration, and iron reduction, which occurred by DFO, DS and Bapta AM administration, and knockdown of tfR1 or IRP2 genes, to investigate the effects of iron metabolism on insulin sensitivity in HSMMs. Intracellular calcium was detected using Fluo-3/AM staining. TfR1 internalization regulated by calcium was detected by GFP-labelled transferrin. Results: Endoplasmic reticulum stress by tunicamycin, thapsigargin, or palmitate evoked insulin resistance and simultaneously increased intracellular iron. Iron chelator, DFO, dramatically prevented ER-stress inducer-induced insulin resistance and iron donor impaired insulin sensitivity in vitro and in vivo through activation of JNK. Among several iron metabolism-related genes, tfR1 plays a predominant role for maintaining iron homeostasis in HSMMs. Treatment of ER-stress inducer positively regulated translocation of tfR1 by intracellular calcium, but protein levels of tfR1 did not change. Iron reduction, by adding iron chelator, calcium chelator, or knockdown of tfR1 or IRP2, dramatically prevented ER-stress-induced insulin resistance through the reduction of iron-overload. Conclusions/Interpretation: The current study shows ER-stress inducer evoked insulin resistance through iron overload. Reduction of intracellular iron, by iron chelator, significantly prevents insulin resistance. Therefore, attempts to block iron overload might be a strategy for preventing insulin resistance and diabetes. Disclosure K. Lee: None. R. Cui: None. D. Kim: None. S. Choi: None. W. Lee: None. Y. Kang: None. T. Kim: None. H. Moon: None. J. Jeon: None. S. Han: None. H. Kim: None.

The TAZ activator 2-butyl-5-methyl-6-(pyridine-3-yl)-3-[2'-(1H-tetrazole-5-yl)-biphenyl-4-ylmethyl]-3H-imidazo[4,5-b]pyridine] (TM-25659) inhibits adipocyte differentiation by interacting with peroxisome proliferator-activated receptor gamma. TM-25659 was previously shown to decrease weight gain in a high fat (HF) diet-induced obesity (DIO) mouse model. However, the fundamental mechanisms underlying the effects of TM-25659 remain unknown. Therefore, we investigated the effects of TM-25659 on skeletal muscle functions in C2 myotubes and C57BL/6J mice. We studied the molecular mechanisms underlying the contribution of TM-25659 to palmitate (PA)-induced insulin resistance in C2 myotubes. TM-25659 improved PA-induced insulin resistance and inflammation in C2 myotubes. In addition, TM-25659 increased FGF21 mRNA expression, protein levels, and FGF21 secretion in C2 myotubes via activation of GCN2 pathways (GCN2-phosphoeIF2α-ATF4 and FGF21). This beneficial effect of TM-25659 was diminished by FGF21 siRNA. C57BL/6J mice were fed a HF diet for 30 weeks. The HF-diet group was randomly divided into two groups for the next 14 days: the HF-diet and HF-diet + TM-25659 groups. The HF diet + TM-25659-treated mice showed improvements in their fasting blood glucose levels, insulin sensitivity, insulin-stimulated Akt phosphorylation, and inflammation, but neither body weight nor food intake was affected. The HF diet + TM-25659-treated mice also exhibited increased expression of both FGF21 mRNA and protein. These data indicate that TM-25659 may be beneficial for treating insulin resistance by inducing FGF21 in models of PA-induced insulin resistance and HF diet-induced insulin resistance.

High levels of plasma free fatty acid are thought to contribute to the loss of pancreatic beta-cells in type 2 diabetes. In particular, saturated fatty acid such as palmitate or stearate can induce apoptosis in cultured beta cells (lipotoxicity). Endoplasmic reticulum stress is a critical mediator of free fatty acid-induced lipotoxicity. Recently, disorders in mitochondrial respiratory metabolism have been linked to lipotoxicity. Since iron is a critical component of respiratory metabolism, this study is initiated to determine whether abnormal iron metabolism is involved in palmitate-induced beta cell death. Immunoblotting analysis showed that treatment of INS-1 beta cells with palmitate reduced the level of transferrin receptor 1 (TfR1), but increased the level of heavy chain ferritin (FTH). In addition, palmitate reduced intracellular labile iron pool. Whereas iron depletion through treatment with iron-chelators deferoxamine or deferasirox augmented palmitate-induced cell death, iron supplementation with ferric chloride, ferrous sulfate, or holo-transferrin significantly protected cells against palmitate-induced death. Furthermore, overexpression of TfR1 reduced palmitate-induced cell death, whereas knockdown of TfR1 augmented cell death. In particular, treatment with deferoxamine increased the level of endoplasmic reticulum (ER) stress markers phospho-PERK, phospho-eIF2α, CHOP and phospho-c-Jun N-terminal kinase. Treatment with chemical chaperone significantly protected cells against deferoxamine-induced apoptosis. Iron supplementation also protected cells against palmitate-induced primary islet death. These data suggest that iron depletion plays an important role in palmitate-induced beta cell death through inducing ER stress. Therefore, attempts to block iron depletion might be able to prevent beta cell loss in type 2 diabetes.

Saturated fatty acids were proposed to activate the NLRP3 inflammasome, a molecular platform that mediates the processing of interleukin (IL)-1β and IL-18. However, the mechanisms underlying the miRNA-mediated regulation of palmitate (PA)-induced inflammasome activation are unclear. We examined the role of miR-132 in PA-induced NLRP3 inflammasome activation in THP-1 cells. To understand the regulatory role of miR-132 in inflammasome activation, we either overexpressed or suppressed miR-132 in THP-1 cells that expressed the NLRP3 inflammasome in response to stimulation by PA. We analyzed the mRNA and protein levels of NLRP3, caspase-1 p10, IL-18, and IL-1β; caspase-1 activity; and IL-1β secretion. The presence of PA activated the NLRP3 inflammasome and increased miR-132 expression. Overexpression of miR-132 reduced caspase-1 p10, IL-18, and IL-1β, while the suppression of miR-132 enhanced inflammasome activation. In addition, miR-132 regulated the mRNA and protein expression of FOXO3, which is a potential target of miR-132 in these cells. FOXO3 suppression by small interfering RNA decreased NLRP3 inflammasome activity stimulated by PA. Knockdown of FOXO3 attenuated NLRP3 inflammasome activation by the miR-132 inhibitor. Based on these findings, we conclude that miR-132 negatively regulates PA-induced NLRP3 inflammasome activation through FOXO3 down-regulation in THP-1 cells.

Background Bitter melon (BM) improves glucose level, lipid homeostasis, and insulin resistance in vivo. However, the preventive mechanism of BM in nonalcoholic fatty liver disease (NAFLD) has not been elucidated yet. Aim & design To determine the protective mechanism of bitter melon extract (BME), we performed experiments in vitro and in vivo. BME were treated palmitate (PA)-administrated HepG2 cells. C57BL/6J mice were divided into two groups: high-fat/high-fructose (HF/HFr) without or with BME supplementation (100 mg/kg body weight). Endoplasmic reticulum (ER) stress, apoptosis, and biochemical markers were then examined by western blot and real-time PCR analyses. Results BME significantly decreased expression levels of ER-stress markers (including phospho-eIF2α, CHOP, and phospho-JNK [Jun N-terminal kinases]) in PA-treated HepG2 cells. BME also significantly decreased the activity of cleaved caspase-3 (a well known apoptotic-induced molecule) and DNA fragmentation. The effect of BME on ER stress-mediated apoptosis in vitro was similarly observed in HF/HFr-fed mice in vivo. BME significantly reduced HF/HFr-induced hepatic triglyceride (TG) and serum alanine aminotransferase (ALT) as markers of hepatic damage in mice. In addition, BME ameliorated HF/HFr-induced serum TG and serum-free fatty acids. Conclusion These data indicate that BME has protective effects against ER stress mediated apoptosis in HepG2 cells as well as in HF/HFr-induced fatty liver of mouse. Therefore, BME might be useful for preventing and treating NAFLD.

Adipokines reportedly affect hepatic gluconeogenesis, and the adipokine visfatin is known to be related to insulin resistance and type 2 diabetes. However, whether visfatin contributes to hepatic gluconeogenesis remains unclear. Visfatin, also known as nicotinamide phosphoribosyltransferase (NAMPT), modulates sirtuin1 (SIRT1) through the regulation of nicotinamide adenine dinucleotide (NAD). Therefore, we investigated the effect of extracellular visfatin on glucose production in HepG2 cells, and evaluated whether extracellular visfatin affects hepatic gluconeogenesis via an NAD+-SIRT1-dependent pathway. Treatment with visfatin significantly increased glucose production and the mRNA expression and protein levels of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) in HepG2 cells in a time- and concentration-dependent manner. Knockdown of SIRT1 had no remarkable effect on the induction of gluconeogenesis by visfatin. Subsequently, we evaluated if extracellular visfatin stimulates the production of gluconeogenic enzymes through the classical protein kinase A (PKA)/cyclic AMP-responsive element (CRE)-binding protein (CREB)-dependent process. The phosphorylation of CREB and PKA increased significantly in HepG2 cells treated with visfatin. Additionally, knockdown of CREB and PKA inhibited visfatin-induced gluconeogenesis in HepG2 cells. In summary, extracellular visfatin modulates glucose production in HepG2 cells through the PKA/CREB pathway, rather than via SIRT1 signaling.

This work was initiated to determine whether toxicity generated through inhibition of mitochondrial fuel metabolism is similar to high glucose/palmitate (HG/PA)-induced glucolipotoxicity. Influx of glucose and free fatty acids into the tricarboxylic acid (TCA) cycle was inhibited by treatment with the pyruvate carboxylase (PC) inhibitor phenylacetic acid (PAA) and carnitine palmitoyl transferase-1 (CPT-1) inhibitor etomoxir (Eto), or knockdown of PC and CPT-1. Treatment of PAA/Eto or knockdown of PC/CPT-1 induced apoptotic death in INS-1 beta cells. Similar to HG/PA treatment, PAA/Eto increased endoplasmic reticulum stress responses but decreased the Akt signal. JNK inhibitor or chemical chaperone was protective against both PAA/Eto- and HG/PA-induced cell death. All attempts to reduce [Ca²⁺](i), stimulate lipid metabolism, and increase the TCA cycle intermediate pool protected PAA/Eto-induced death as well as HG/PA-induced death. These data suggest that signals induced from impaired mitochondrial fuel metabolism play a critical role in HG/PA-induced glucolipotoxicity.

The circadian clock organizes the physiology and behavior of organisms to their daily environmental rhythms. The central circadian timekeeping mechanism in eukaryotic cells is the transcriptional-translational feedback loop (TTFL). In the Drosophila TTFL, the transcription factors CLOCK (CLK) and CYCLE (CYC) play crucial roles in activating expression of core clock genes and clock-controlled genes. Many signaling pathways converge on the CLK/CYC complex and regulate its activity to fine-tune the cellular oscillator to environmental time cues. We aimed to identify factors that regulate CLK by performing tandem affinity purification combined with mass spectrometry using Drosophila S2 cells that stably express HA/FLAG-tagged CLK and V5-tagged CYC. We identified SNF4Aγ, a homolog of mammalian AMP-activated protein kinase γ (AMPKγ), as a factor that copurified with HA/FLAG-tagged CLK. The AMPK holoenzyme composed of a catalytic subunit AMPKα and two regulatory subunits, AMPKβ and AMPKγ, directly phosphorylated purified CLK in vitro Locomotor behavior analysis in Drosophila revealed that knockdown of each AMPK subunit in pacemaker neurons induced arrhythmicity and long periods. Knockdown of AMPKβ reduced CLK levels in pacemaker neurons, and thereby reduced pre-mRNA and protein levels of CLK downstream core clock genes, such as period and vrille Finally, overexpression of CLK reversed the long-period phenotype that resulted from AMPKβ knockdown. Thus, we conclude that AMPK, a central regulator of cellular energy metabolism, regulates the Drosophila circadian clock by stabilizing CLK and activating CLK/CYC-dependent transcription.SIGNIFICANCE STATEMENT Regulation of the circadian transcription factors CLK and CYC is fundamental to synchronize the core clock with environmental changes. Here, we show that the AMPKγ subunit of AMPK, a central regulator of cellular metabolism, copurifies with the CLK/CYC complex in Drosophila S2 cells. Furthermore, the AMPK holoenzyme directly phosphorylates CLK in vitro This study demonstrates that AMPK activity regulates the core clock in Drosophila by activating CLK, which enhances circadian transcription. In mammals, AMPK affects the core clock by downregulating circadian repressor proteins. It is intriguing to note that AMPK activity is required for core clock regulation through circadian transcription enhancement, whereas the target of AMPK action is different in Drosophila and mammals (positive vs negative element, respectively).

Several association studies have implicated the PARK2 gene that encodes parkin – the key molecule orchestrating the mitochondrial quality control system – as a candidate susceptibility gene for diabetes. A total of 7551 unrelated Korean KARE cohort subjects were analyzed to investigate the association between the PARK2 single nucleotide polymorphism (SNP) and quantitative glycemic traits. Two SNPs, rs10455889 and rs9365294, were significantly associated with fasting plasma glucose level ( p = ∼1.2 × 10 −4 ) and insulin secretion indices ( p = ∼7.4 × 10 −5 ) in male KARE subjects. Parkin was expressed predominantly in the rat pancreatic islets. Downregulation of the Park2 gene in rat INS-1 β-cells resulted in a significant decrease in the glucose-stimulated insulin secretion, intracellular insulin gene expression, and intracellular ATP level. The Park2 -depleted β-cells also exhibited increased mitochondrial fragmentation and ROS production and decreased mitochondrial membrane potential. Both population-based statistical evaluation and experimental evidence demonstrated a fundamental role of the PARK2 gene in the maintenance of β-cell function.

Beta cell loss and insulin resistance play roles in the pathogenesis of type 2 diabetes. Elevated levels of free fatty acids in plasma might contribute to the loss of beta cells. The objective of this study was to find a chemical that could protect against palmitate-induced beta cell death and investigate whether such chemical could improve hyperglycemia in mouse model of type 2 diabetes. Sodium fluorocitrate (SFC), an aconitase inhibitor, was found to be strongly and specifically protective against palmitate-induced INS-1 beta cell death. However, the protective effect of SFC on palmitate-induced cell death was not likely to be due to its inhibitory activity for aconitase since inhibition or knockdown of aconitase failed to protect against palmitate-induced cell death. Since SFC inhibited the uptake of palmitate into INS-1 cells, reduced metabolism of fatty acids was thought to be involved in SFC’s protective effect. Ten weeks of treatment with SFC in db/db diabetic mice reduced glucose level but remarkably increased insulin level in the plasma. SFC improved impairment of glucose-stimulated insulin release and also reduced the loss of beta cells in db/db mice. Conclusively, SFC possessed protective effect against palmitate-induced lipotoxicity and improved hyperglycemia in mouse model of type 2 diabetes.

Individuals with non-alcoholic fatty liver disease (NAFLD) and type 2 diabetes (T2D) induced by high calorie western diet are characterized by enhanced lipogenesis and gluconeogenesis in the liver. Stimulation of reductive amination may shift tricarboxylic acid cycle metabolism for lipogenesis and gluconeogenesis toward glutamate synthesis with increase of NAD+/NADH ratio and thus, ameliorate high calorie diet-induced fatty liver and hyperglycemia. Stimulation of reductive amination through glutamate dehydrogenase (GDH) activator 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) reduced both de novo lipogenesis and gluconeogenesis but increased the activities of sirtuins and AMP-activated kinase in primary hepatocytes. Long-term BCH treatment improved most metabolic alterations induced by high fat/high fructose (HF/HFr) diet in C57BL/6J mice. BCH prevented HF/HFr-induced fat accumulation and activation of stress/inflammation signals such as phospho-JNK, phospho-PERK, phospho-p38 and phospho-NFκB in liver tissues. Furthermore, BCH treatment reduced the expression levels of inflammatory cytokines such as TNF-α and IL-1β in HF/HFr-fed mouse liver. BCH also reduced liver collagen and plasma levels of alanine transaminase and aspartate transaminase. On the other hand, BCH significantly improved fasting hyperglycemia and glucose tolerance in HF/HFr-fed mice. In conclusion, stimulation of reductive amination through GDH activation can be used as a strategy to prevent high calorie western diet-induced NAFLD and T2D.

Deregulation of glutamate homeostasis is associated with degenerative neurological disorders. Glutamate dehydrogenase (GDH) is important for glutamate metabolism and plays a central role in expanding the pool of tricarboxylic acid (TCA) cycle intermediate alpha-ketoglutarate (α-KG), which improves overall bioenergetics. Under high energy demand, maintenance of ATP production results in functionally active mitochondria. Here, we tested whether the modulation of GDH activity can rescue ischemia/reperfusion-induced neuronal death in an in vivo mouse model of middle artery occlusion and an in vitro oxygen/glucose depletion model. Iodoacetate, an inhibitor of glycolysis, was also used in a model of energy failure, remarkably depleting ATP and α-KG. To stimulate GDH activity, the GDH activator 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid and potential activator beta-lapachone were used. The GDH activators restored α-KG and ATP levels in the injury models and provided potent neuroprotection. We also found that beta-lapachone increased glutamate utilization, accompanied by a reduction in extracellular glutamate. Thus, our hypothesis that mitochondrial GDH activators increase α-KG production as an alternative energy source for use in the TCA cycle under energy-depleted conditions was confirmed. Our results suggest that increasing GDH-mediated glutamate oxidation represents a new therapeutic intervention for neurodegenerative disorders, including stoke.

Prolonged exposure to high glucose (HG) and palmitate (PA) results in increased ER stress and subsequently induces β-cell apoptosis. Exendin-4, a glucagon-like peptide-1 agonist, is known to protect β cells from toxicity induced by cytokines, HG, or fatty acids by reducing ER stress. However, the detailed molecular mechanisms for this protective effect are still not known. In this study, we investigated the role of exendin-4 in the inhibition of glucolipotoxicity-induced ER stress and β-cell apoptosis. Exendin-4 treatment protected INS-1 β cells from apoptosis in response to HG/PA (25 mM glucose+400 μM PA). HG/PA treatment increased cleaved caspase-3 and induced ER stress maker proteins such as PERK (EIF2AK3), ATF6, and phosphorylated forms of PERK, eIF2α, IRE1α (ERN1), and JNK (MAPK8), and these increases were significantly inhibited by exendin-4 treatment. HG/PA treatment of INS-1 cells increased SREBP1 (SREBF1) protein and induced its nuclear translocation and subsequently increased C/EBPβ (CEBPB) protein and its nuclear translocation. Exendin-4 treatment attenuated this increase. Knockdown ofSREBP1creduced the activation ofC/EBPβand also blocked the expression of ER stress markers induced by HG/PA treatment. Our results indicate that exendin-4 inhibits the activation of SREBP1c and C/EBPβ, which, in turn, may reduce glucolipotoxicity-induced ER stress and β-cell apoptosis.

Free fatty acids (FFAs) lead to the activation of inflammatory pathways related to the induction of insulin resistance. Visfatin is known to play a role in obesity-related metabolic diseases and inflammatory conditions. Here, the role of visfatin in FFA-induced inflammation was investigated in hepatocytes. The following factors were examined: (1) the protein and messenger RNA (mRNA) expression of visfatin in the liver tissue of insulin-resistant rats and in (2) in HepG2 cells treated with palmitate, (3) the palmitate-induced mRNA expression and protein synthesis of interleukin-6 and tumor necrosis factor-α in HepG2 cells transfected with visfatin-specific small interfering RNA, and (4) the expression of visfatin in HepG2 cells treated with a nuclear factor-κB (NF-κB) inhibitor (SN50) and infected with Ad-IκBα. The protein and mRNA levels of visfatin were significantly higher in insulin-resistant rat liver tissue compared with the control group. Visfatin expression and protein synthesis significantly increased in HepG2 cells treated with palmitate in a time- and concentration-dependent manner. Visfatin-specific small interfering RNA significantly decreased the palmitate-induced mRNA expression and protein synthesis of interleukin-6 and tumor necrosis factor-α. A NF-κB inhibitor induced the downregulation of visfatin in HepG2 cells following treatment with palmitate. HepG2 cells infected with Ad-IκBα showed decreased expression of visfatin following treatment with palmitate. The expression of visfatin is closely associated with the expression of proinflammatory cytokines in FFA-induced inflammation and is significantly decreased by NF-κB inhibition in HepG2 cells. Visfatin may play a role in FFA-induced inflammation in hepatocytes through the NF-κB pathway.