PP2Ac knockdown attenuates lipotoxicity­induced pancreatic ß­cell dysfunction and apoptosis.

Protein phosphatase 2A (PP2A) is one of the most common serine/threonine phosphatases in mammalian cells, and it primarily functions to regulate cell signaling, glycolipid metabolism and apoptosis. The catalytic subunit of PP2A (PP2Ac) plays an important role in the functions of the protein. However, there are few reports on the regulatory role of PP2Ac in pancreatic ß-cells under lipotoxic conditions. In the present study, mouse insulinoma 6 (MIN6) pancreatic cells were transfected with short hairpin RNAs to generate PP2Ac knockdown cells and incubated with palmitate (PA) to establish a lipotoxicity model. Serine/threonine phosphatase assay system, Cell Counting Kit-8, flow cytometry, enzyme-linked immunosorbent assay and western blotting were used to measure PP2A activity, cell viability, apoptosis, oxidative stress and insulin secretion in the cells. In addition, a mouse model of lipotoxicity was established with a high-fat diet (HFD) and the knockdown of PP2Ac using adeno-associated viruses to interfere with PP2Ac expression in the pancreatic tissues. The activity of PP2A in the mouse pancreatic tissue and the serum insulin level were measured. Furthermore, the proliferation of mouse pancreatic ß-cells was assessed using pancreatic tissue immunofluorescence. PP2Ac knockdown inhibited lipotoxicity-induced PP2A hyperactivation, increased the resistance of pancreatic ß-cells to lipotoxicity and attenuated PA-induced apoptosis in MIN6 cells. It also protected the endoplasmic reticulum and mitochondria, and ameliorated insulin secretion. The results of mRNA sequencing and western blotting analysis suggested that the protective effects of PP2Ac knockdown in MIN6 cells may be mediated via the MAPK pathway. Moreover, the results of the animal experiments suggested that specific knockdown of pancreatic PP2Ac effectively attenuated HFD-induced insulin resistance and reduced the compensatory proliferation of pancreatic ß-cells in mice. In summary, the present study revealed the effects of interfering with PP2Ac gene expression on pancreatic ß-cells in vivo and in vitro and the underlying mechanisms, which may provide insights for the treatment of type 2 diabetes mellitus in the clinic.

Bridging the Gap between Charge Storage Site and Transportation Pathway in Molecular-Cage-Based Flexible Electrodes.

Porous materials have been widely applied for supercapacitors; however, the relationship between the electrochemical behaviors and the spatial structures has rarely been discussed before. Herein, we report a series of porous coordination cage (PCC) flexible supercapacitors with tunable three-dimensional (3D) cavities and redox centers. PCCs exhibit excellent capacitor performances with a superior molecular capacitance of 2510 F mmol-1, high areal capacitances of 250 mF cm-2, and unique cycle stability. The electrochemical behavior of PCCs is dictated by the size, type, and open-close state of the cavities. Both the charge binding site and the charge transportation pathway are unambiguously elucidated for PCC supercapacitors. These findings provide central theoretical support for the "structure-property relationship" for designing powerful electrode materials for flexible energy storage devices.

Hepatic IDH2 regulates glycolysis and gluconeogenesis.

BACKGROUND AND AIMS: The liver plays a central role in controlling glucose and lipid metabolism. IDH2, a mitochondrial protein, controls TCA cycle flux. However, its role in regulating metabolism in obesity is still unclear. This study intends to investigate the impact of hepatic IDH2 expression on overnutrition-regulated glucose and lipid metabolism. METHODS: Hepatic IDH2 was knocked-out in mice by the approach of CRISPR-Cas9. Mice were subjected to starvation and refeeding for hepatic glucose and lipid studies in vivo. Primary hepatocytes and mouse normal liver cell line, AML12 cells were used for experiments in vitro. RESULTS: This study found that IDH2 protein levels were elevated in the livers of obese people and mice with high-fat diet consumption or hepatic steatosis. Liver IDH2-deletion mice (IDH2LKO) were resistant to high-fat diet-induced body weight gain, with lower serum glucose and TG levels, increased insulin sensitivity, and higher FGF21 secretion, despite the higher TG content in the liver. Consistently, overexpression of IDH2 in hepatocytes promoted gluconeogenesis and enhanced glycogenesis. By performing mass spectrometry and proteomics analyses, we further demonstrated that IDH2-deficiency in hepatocytes accelerated ATP production by increasing forward TCA cycle flux, thus promoting glycolysis pathway and decreasing glycogen synthesis at refeeding state, and inhibiting hepatic gluconeogenesis, increasing ß-oxidation during starvation. Moreover, experiments in vivo demonstrated that IDH2-knockout might not exacerbate hepatic inflammatory responses in the NASH model. CONCLUSIONS: Elevated hepatic IDH2 under over-nutrition state contributes to elevated gluconeogenesis and glycogen synthesis. Inhibition of IDH2 in the liver could be a potential therapeutic target for obesity and diabetes.

Sigma­1 receptor overexpression promotes proliferation and ameliorates cell apoptosis in ß­cells.

Sigma­1 receptor (Sig­1R) is a class of orphan receptors, the potential role of which in pancreatic islet cells remains poorly understood. The present study aimed to investigate the role of Sig­1R in islet ß­cell proliferation and examine the effects of Sig­1R on islet ß­cell injury under lipotoxic conditions. Sig­1R­overexpressing MIN6 cells were generated by lentiviral vector transfection. The effect of Sig­1R overexpression on cell proliferation detected by EdU staining, cell cycle progression by propidium iodide (PI), apoptosis by Annexin V­APC/PI, mitochondrial membrane potential by Mitolite Red and cytoplasmic Ca2+ levelsby Fura­2/AM in islet ß­cells were measured by flow cytometry. Western blot analysis was used to measure protein expression levels of endoplasmic reticulum (ER) stress markers glucose­regulated protein 78 and C/EBP homologous protein, mitochondrial apoptotic proteins Bcl­2­associated X and Bcl­2 and cytochrome c. In addition, ATP levels and insulin secretion were separately measured using ATP Assay and mouse insulin ELISA. Mitochondria­associated ER membrane (MAM) structures in MIN6 cells were then detected using transmission electron microscopy. Protein disulfide isomerase expression and possible colocalization between inositol 1,4,5­trisphosphate receptor and voltage­dependent anion channel 1 were examined using immunofluorescence. Sig­1R overexpression was found to promote ß­cell proliferation by accelerating cell cycle progression. Furthermore, Sig­1R overexpression ameliorated the apoptosis rate whilst impairing insulin secretion induced by palmitic acid by relieving ER stress and mitochondrial dysfunction in MIN6 cells. Sig­1R overexpression also promoted Ca2+ transport between mitochondria and ER by increasing the quantity of ER adjacent to mitochondria in the 50­nm range. It was concluded that Sig­1R overexpression conferred protective effects on ß­cells against lipotoxicity as a result of the promotion of cell proliferation and inhibition of ER stress and oxidative stress, by regulating the structure of MAM.

The effects of extreme temperatures on emergency room visits-a population-based analysis by age, sex, and comorbidity.

This study evaluated the effect of extreme temperatures on events requiring emergency room visits (ERVs) for hypertensive disease, ischemic heart disease (IHD), cerebrovascular disease, and chronic kidney disease (CKD) for population stratified by sex and age living in Taiwan's metropolitan city from 2000 to 2014. The distributed lag non-linear model was adopted to examine the association between ambient temperature and area-age-sex-disease-specific ERVs for a population aged 40 years and above. The reference temperature was defined by a percentile value to describe the temperature in each city. Area-age-sex-disease-specific relative risk (RR) and 95% confidence intervals (CI) were estimated in association with extreme high (99th percentile) and low (5th percentile) temperatures. Temperature-related ERV risks varied by area, age, sex, and disease. Patients with CKD tend to have comorbidities with hypertensive disease. All study populations with hypertensive disease have significant risk associations with extreme low temperatures with the highest RR of 2.64 (95% CI: 2.08, 3.36) appearing in New Taipei City. The risk of IHD was significantly associated with extreme high temperature for male subpopulation aged 40-64 years. A less significant association was observed between the risks of cerebrovascular disease with extreme temperature. The risk of CKD was most significantly associated with extreme high temperature especially for a subpopulation aged 40-64 years. All study subpopulations with hypertensive disease have significant risk associations with extreme low temperature. Male subpopulations were more vulnerable to extreme temperatures, especially for those aged 40-64 years.

Nr2e1 deficiency aggravates insulin resistance and chronic inflammation of visceral adipose tissues in a diet-induced obese mice model.

AIMS: To investigate the nuclear receptor subfamily 2 group E member 1 (Nr2e1) expression in adipose tissues of obese mice and assess the role of Nr2e1 in insulin resistance and chronic inflammation of the adipose tissues. MAIN METHODS: An obese model was established in Nr2e1 knockout (KO) mice and their wild type (WT) littermates through a long-term high-fat diet (HFD) feeding regime. The epididymal fat weight, body weight, and daily food intake were recorded. The blood lipid profile, blood inflammatory factors, and the levels of fasting blood glucose (FBG) and fasting insulin were determined. We estimated insulin resistance by the homeostasis model assessment (HOMA). The expression of inflammatory factors and F4/80 was examined by polymerase chain reaction (PCR) and western blotting to assess adipose tissues inflammation. We also determined the molecules of insulin signaling and the nuclear factor kappa B (NF-κB) pathway by western blotting. KEY FINDINGS: The Nr2e1 expression was upregulated in WT obese mice when compared with that in control mice. Despite a lower body weight and epididymal fat mass in Nr2e1-/- mice, these rats showed increased inflammatory cytokines secretion, more pronounced hyperlipidemia, and impaired insulin sensitivity after HFD treatment. Further investigation revealed that Nr2e1 deletion affected the expression of insulin signaling and NF-κB pathway-related molecules in visceral adipose tissues. SIGNIFICANCE: Nr2e1 may act as a potential target to improve insulin sensitivity and inflammation in obesity and related complications.

Sigma receptor knockdown augments dysfunction and apoptosis of beta cells induced by palmitate.

Sigma-1 receptor (Sig-1R) is located in the endoplasmic reticulum (ER) and clustered on the mitochondria related endoplasmic membranes, which are involved in the regulation of nervous system disease. Here, we designed Sig-1R silence MIN6 cells and studied the influence of Sig-1R silence on beta cells. We showed Sig-1R inactivation in MIN6 cells could not only decrease cell proliferation but also inhibit cell cycle, and this inhibitory effect on cell cycle might be achieved by regulating the FoxM1/Plk1/Cenpa pathway. Moreover, Sig-1R deficiency increased MIN6 cells sensitivity to lipotoxicity, exaggerated palmitate (PA)-induced apoptosis, and impaired insulin secretion. On the other hand, ER chaperone GRP78 and ER proapoptotic molecules CHOP increased in Sig-1R knockdown MIN6 cells. The ATP level decreased and reactive oxygen species (ROS) increased in this kind of cells. Furthermore not only GRP78 and CHOP levels, but also ATP and ROS levels changed more in Sig-1R silence cells after cultured with PA. Therefore, Sig-1R deficiency exaggerated PA induced beta cells apoptosis by aggravating ER stress and mitochondrial dysfunction. Together, our study showed that Sig-1R might influence the proliferation, apoptosis, and function of beta cells.