Protective effect of empagliflozin against palmitate-induced lipotoxicity through AMPK in H9c2 cells.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors have recently emerged as novel cardioprotective agents. However, their direct impact on cardiomyocyte injury is yet to be studied. In this work, we investigate the underlying molecular mechanisms of empagliflozin (EMPA), an SGLT2 inhibitor, in mitigating palmitate (PA)-induced cardiomyocyte injury in H9c2 cells. We found that EMPA significantly attenuated PA-induced impairments in insulin sensitivity, ER stress, inflammatory cytokine gene expression, and cellular apoptosis. Additionally, EMPA elevated AMP levels, activated the AMPK pathway, and increased carnitine palmitoyl transferase1 (CPT1) gene expression, which collectively enhanced fatty acid oxidation and reduced stress signals. This study reveals a novel mechanism of EMPA's protective effects against PA-induced cardiomyocyte injury, providing new therapeutic insights into EMPA as a cardioprotective agent.

[Biological Function of CysR Domain of ADAMTS13].

OBJECTIVE: To investigate the biological function of Cysteine rich (CysR) domain of a disintegrin and metalloprotease with thrombospondin type 1 repeats-13 (ADAMTS13) on cleavage of von Willebrand factor (vWF) and provide experimental evidence for exploring the pathogenesis of thrombotic thrombocytopenic purpura (TTP). METHODS: The six amino acids (EDGTLS) in ADAMTS13 CysR domain were point mutated one by one, and the mutant ADAMTS13 proteins were expressed and purified. The cleavage products of vWF polymer by wild-type or mutant ADAMTS13 under denaturing condition or shear stress were separated by 1% SeaKem HGT agarose gel and detected by Western blot. RESULTS: The mutant ADAMTS13 plasmids (M1: Glu515Ala; M2: Asp516Ala; M3: Gly517Ala; M4: Thr518Ala; M5: Leu519Ala; M6: Ser520Ala) were successfully constructed and the proteins of wild-type and mutant ADAMTS13 were purified. Wild-type ADAMTS13 almost completely cleaved the vWF polymer under denaturing condition, while the cleavage activity of M1 mutant was significantly reduced in the same condition (P<0.01). The cleavage activity of M1 mutant of ADAMTS13 was also significantly reduced compared with that of the wild-type under shear stress (P<0.01). The activity of M1 mutant to cleave the FRETS-vWF73 was dramatically reduced compared with that of wild-type ADAMTS13. However, the binding ability of M1 mutant to vWF was similar with that of wild-type ADAMTS13. CONCLUSION: The CysR domain of ADAMTS13 plays an important role in the digestion of vWF under denaturing condition and shear stress. The Glu515 amino acid residue might be an important site for substrate recognition.

CUDC-101 enhances the chemosensitivity of gemcitabine-treated lymphoma cells.

BACKGROUND: The metastasis and recurrence of Non-Hodgkin's lymphoma (NHL) is a major cause of morbidity and mortality. Recent work suggests that drugs capable of targeting epigenetic regulatory mechanisms may be well suited to the treatment of such disease progression. METHODS: This study was thus designed to evaluate the ability of the novel histone deacetylase (HDAC) inhibitor CUDC-101 to synergize with gemcitabine in order to kill human HUT78 and Pfeiffer NHL cells. To that end, we analyzed the viability of these NHL cells via CCK-8 assay, while the incidence of apoptosis among treated cells was evaluated via Annexin V-FITC/PI staining and by the Western blotting-mediated evaluation of proteins associate with apoptosis and related signaling pathways. RESULTS: We found that CUDC-101 and gemcitabine interacted synergistically to reduce NHL cell viability and to induce the apoptotic death of these cells via the EGFR/ PI3K/Akt and Erk pathways, which were regulated by HDAC signaling pathways. CONCLUSION: Together, our results highlight the anti-cancer properties of CUDC-101 alone or in combination with gemcitabine as an approach to inducing the apoptotic death of lymphoma cells in vitro, while also offering insight into the underlying molecular mechanisms governing this activity.

Sodium fluorocitrate having inhibitory effect on fatty acid uptake ameliorates high fat diet-induced non-alcoholic fatty liver disease in C57BL/6J mice.

Non-alcoholic fatty liver disease (NAFLD) is excessive fat build-up in the liver without alcohol consumption and includes hepatic inflammation and damage. Excessive influx of fatty acids to liver from circulation is thought to be a pathogenic cause for the development of NAFLD. Thus, inhibition of fatty acid intake into hepatocyte would be a maneuver for protection from high fat diet (HFD)-induced NAFLD. This study was initiated to determine whether sodium fluorocitrate (SFC) as a fatty acid uptake inhibitor could prevent palmitate-induced lipotoxicity in hepatocytes and protect the mice from HFD-induced NAFLD. SFC significantly inhibited the cellular uptake of palmitate in HepG2 hepatocytes, and thus prevented palmitate-induced fat accumulation and death in these cells. Single treatment with SFC reduced fasting-induced hepatic steatosis in C57BL/6J mice. Concurrent treatment with SFC for 15 weeks in HFD-fed C57BL/6J mice prevented HFD-induced fat accumulation and stress/inflammatory signal activation in the liver. SFC restored HFD-induced increased levels of serum alanine aminotransferase and aspartate aminotransferases as hepatic injury markers in these mice. SFC treatment also improved HFD-induced hepatic insulin resistance, and thus ameliorated HFD-induced hyperglycemia. In conclusion, inhibition of fatty acid mobilization into liver through SFC treatment can be a strategy to protect from HFD-induced NAFLD.

Iron overload by transferrin receptor protein 1 regulation plays an important role in palmitate-induced insulin resistance in human skeletal muscle cells.

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.

Bitter melon extract ameliorates palmitate-induced apoptosis via inhibition of endoplasmic reticulum stress in HepG2 cells and high-fat/high-fructose-diet-induced fatty liver.

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.