Lack of Nr2e1 expression in hepatocytes impaired cell survival and aggravated palmitate-induced oxidative stress.

PURPOSE: Nuclear receptor subfamily 2 group E member 1 (Nr2e1) has been regarded as an essential regulator in neural stem cells. However, its function is still not clear in hepatocytes. This study aimed to clarify the effects of Nr2e1-deficiency in hepatocytes in lipotoxic conditions. MATERIALS/METHODS: Nr2e1-knockdown AML12 cells were generated by lentiviral vector transfection. The influences of Nr2e1-deficiency on hepatocyte survival were determined by cell cycle progression and cell apoptosis rate using flow cytometry. Real-time quantitative PCR and Western blot were used to examine the genes and protein expression related to apoptosis, lipid metabolism, and oxidative stress. Meanwhile, RNA sequencing was adopted in liver samples from Nr2e1-knockout (Nr2e1-KO) mice. RESULTS: Nr2e1 expression was observed with a significant decrease in AML12 cells after palmitic acid-stimulation. Knockdown of Nr2e1 in AML12 cells resulted in increased sensitivity to lipotoxicity, evidenced by a partial G0/G1 cell-cycle arrest and higher rates of cell apoptosis. Moreover, Nr2e1-knockdown AML12 cells presented increased gene expressions relative to lipid synthesis but decreased levels of ß-oxidation related genes. Lack of Nr2e1 augmented palmitate-induced oxidative stress in hepatocytes. In vivo, differential genes in Nr2e1-KO mice liver were enriched in pathways associated with liver regeneration and cell proliferation. CONCLUSIONS: This study indicated that hepatocytes lacking Nr2e1 were more susceptible to lipotoxic-mediated damage. Nr2e1 may serve as a potential target for the development of novel therapies for lipotoxicity-induced liver injury.

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.

FUNDC1 modulates mitochondrial defects and pancreatic ß-cell dysfunction under lipotoxicity.

Insulin resistance and many metabolic disorders are causally linked to mitochondrial dysfunction or defective mitochondrial quality control. Mitophagy is a highly selective mechanism that recognizes and removes damaged mitochondria to maintain mitochondrial homeostasis. Here, we addressed the potential role of FUNDC1, a mediator of mitophagy, in pancreatic ß-cell dysfunction under lipotoxicity. In pancreatic MIN6 cells, FUNDC1 deficiency aggravated palmitate-induced mitochondrial dysfunction, which led to cell death and insulin insensitivity. Interestingly, FUNDC1 overexpression prevented these cellular harms brought on by palmitate. In mice models, pancreatic-specific FUNDC1 overexpression alleviated high-fat diet (HFD)-induced insulin resistance and obesity. Mechanistically, pancreatic-specific overexpression of FUNDC1 ameliorated mitochondrial defects and endoplasmic reticulum (ER) stress upon HFD. Our research indicates that FUNDC1 plays an essential role in apoptosis and dysfunction of pancreatic ß-cells via modulating lipotoxicity-induced mitochondrial defects.

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.

Neuroprotective Mechanism of Icariin on Hypoxic Ischemic Brain Damage in Neonatal Mice.

Objective: Our previous results showed that icariin (ICA) could inhibit apoptosis and provide neuroprotection against hypoxic-ischemic brain damage (HIBD) in neonatal mice, but the specific mechanism of its neuroprotective effect remains unknown. This study aims at exploring whether ICA plays a neuroprotective role in apoptosis inhibition by regulating autophagy through the estrogen receptor α (ERα)/estrogen receptor ß (ERß) pathway in neonatal mice with HIBD. Methods: A neonatal mouse model of HIBD was constructed in vivo, and an oxygen and glucose deprivation (OGD) model in HT22 cells from the hippocampal neuronal system was constructed in vitro. The effects of ICA pretreatment on autophagy and the expression of ERα and ERß were detected in vitro and in vivo, respectively. ICA pretreatment was also supplemented with the autophagy inhibitor 3-methyladenine (3-MA), ERα inhibitor methylpiperidino pyrazole (MPP), and ERß inhibitor 4-(2-phenyl-5,7-bis (trifluoromethyl) pyrazolo [1,5-a] pyramidin-3-yl) phenol (PHTPP) to further detect whether ICA pretreatment can activate the ERα/ERß pathway to promote autophagy and reduce HIBD-induced apoptosis to play a neuroprotective role against HIBD in neonatal mice. Results: ICA pretreatment significantly promoted autophagy in HIBD mice. Treatment with 3-MA significantly inhibited the increase in autophagy induced by ICA pretreatment, reversed the neuroprotective effect of ICA pretreatment, and promoted apoptosis. Moreover, ICA pretreatment significantly increased the expression levels of the ERα and ERß proteins in HIBD newborn mice. Both MPP and PHTPP administration significantly inhibited the expression levels of the ERα and ERß proteins activated by ICA pretreatment, reversed the neuroprotective effects of ICA pretreatment, inhibited the increase in autophagy induced by ICA pretreatment, and promoted apoptosis. Conclusion: ICA pretreatment may promote autophagy by activating the ERα and ERß pathways, thus reducing the apoptosis induced by HIBD and exerting a neuroprotective effect on neonatal mice with HIBD.

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.

Inflammatory state and autism-like behavioral phenotype of offspring induced by maternal exposure to low-dose chemical mixtures during pregnancy in mice.

To investigate the effects of maternal exposure to 13 chemicals mixture (CM) during pregnancy on pregnancy outcome and health status of maternal/offspring mice. C57BL/6 pregnant mice were given drinking water containing carbaryl dimethoate glyphosate methomyl methyl parathion triadimefon aspartame sodium benzoate calcium disodium ethylene diamine tetra-acetate ethylparaben butylparaben bisphenol A and acacia gum The effects of CM exposure on pregnancy outcome, health status of dams/offspring, levels of circulating inflammatory cytokines in dams/offspring and emotional related behaviors of offspring were evaluated. CM exposure during pregnancy had no significant effect on pregnancy outcome, liver function, body weight of the dams in late pregnancy and uterine/ovarian weight after delivery, however, it led to an increase in maternal serum IFN-γ level （<0.05）. CM exposure during pregnancy had no significant effect on the liver function of offspring, but increased the serum IFN-γ, prefrontal cortex IFN-γ, and TNF-α and hippocampus IFN-γ levels in the offspring（all <0.01）. In addition, the offspring of CM group showed significant abnormal emotion-related (autism-like) behaviors in adulthood, especially in male offspring. Low dose CM exposure during pregnancy may induce inflammation status in dams/offspring, and lead to autism-like behaviors in offspring, indicating the potential effects of low dose CM exposure on human maternal and infant health.

Small intestinal flora graft alters fecal flora, stool, cytokines and mood status in healthy mice.

Fecal microbiota transplantation is widely used. Large intestinal microbiota (LIM) is more similar to fecal microbiota than small intestinal microbiota (SIM). The SIM communities are very different from those of LIM. Therefore, SIM transplantation (SIMT) and LIM transplantation (LIMT) might exert different influences. Here, healthy adult male C57Bl/6 mice received intragastric SIMT, LIMT, or sterile PBS administration. Microbiota graft samples were collected from small/large intestine of healthy mice of the same age, sex, and strain background. Compared with PBS treatment, SIMT increased pellet number, stool wet weight, and stool water percentage; induced a fecal microbiota profile shift toward the microbial composition of the SIM graft; induced a systemic anti-inflammatory cytokines profile; and ameliorated depressive-like behaviors in recipients. LIMT, however, induced merely a slight alteration in fecal microbial composition and no significant influence on the other aspects. In sum, SIMT, rather than LIMT, affected defecation features, fecal microbial composition, cytokines profile, and depressive-like behaviors in healthy mice. This study reveals the different effects of SIMT and LIMT, providing an interesting clue for further researches involving gut microbial composition change.

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.

Neonatal LPS exposure reduces ATP8A2 level in the prefrontal cortex in mice via increasing IFN-γ level.

Neonatal lipopolysaccharide (LPS) exposure can cause depressive-like behaviors in rodents involving elevated interferon (IFN)-γ. Studies have linked down-regulation of prefrontal cortex (PFC) ATPase phospholipid transporting 8A2(ATP8A2) expression to depressive-like behaviors. In non-neuronal cells, IFN-γ could reduce ATP8A2 expression. Therefore, we hypothesized that neonatal LPS exposure might induce PFC ATP8A2 down-regulation by increasing the IFN-γ level. Here, C57BL6/J mice of both sexes received 3-dose-injections of LPS (50 µg/kg body weight, i.p.) on postnatal day (PND)5, PND7, and PND9. LPS-treated mice showed a transiently decreased PFC ATP8A2 expression indicated by western blot results. Moreover, a significant negative correlation of PFC ATP8A2 expression was found with the IFN-γ level. Using neutralizing mAb, IFN-γ was identified as the key mediator of LPS-induced PFC ATP8A2 decrease indicated by western blot and immunofluorescence results. In sum, neonatal LPS exposure reduced ATP8A2 level in PFC in mice via increasing IFN-γ level. This finding may help further understand the mechanism underlying LPS-induced impairments in brain development and function.

Nr2e1 ablation impairs liver glucolipid metabolism and induces inflammation, high-fat diets amplify the damage.

Nonalcoholic fatty liver disease (NAFLD) is a common and complex metabolic disorder. Despite the widespread concern, there are still few effective treatments except lifestyle interventions. Nuclear receptor subfamily 2 group E member 1 (Nr2e1) is a transcription factor which regulates many biological processes, including development, growth, and differentiation of nerve cells. However, its specific function in hepatocyte is still unknown. In the present study, we found that the expression of Nr2e1 decreased in the livers of high-fat diet-fed mice. We generated Nr2e1 knockout (KO) mice and studied whether Nr2e1 ablation was related to NAFLD. We found that typical pathological features of NAFLD, including insulin resistance, hepatic steatosis, and inflammation, were present in Nr2e1-KO mice or high-fat diet-induced mice models. In conclusion, Nr2e1 ablation promotes liver steatosis and systemic insulin resistance. Nr2e1 may play a protective role in the formation of NAFLD and may serve as a worthy therapeutic target for NAFLD.

Astroglia overexpressing heme oxygenase-1 predispose co-cultured PC12 cells to oxidative injury.

The mechanisms responsible for the progressive degeneration of dopaminergic neurons and pathologic iron deposition in the substantia nigra pars compacta of patients with Parkinson's disease (PD) remain unclear. Heme oxygenase-1 (HO-1), the rate-limiting enzyme in the oxidative degradation of heme to ferrous iron, carbon monoxide, and biliverdin, is upregulated in affected PD astroglia and may contribute to abnormal mitochondrial iron sequestration in these cells. To determine whether glial HO-1 hyper-expression is toxic to neuronal compartments, we co-cultured dopaminergic PC12 cells atop monolayers of human (h) HO-1 transfected, sham-transfected, or non-transfected primary rat astroglia. We observed that PC12 cells grown atop hHO-1 transfected astrocytes, but not the astroglia themselves, were significantly more susceptible to dopamine (1 microM) + H(2)O(2) (1 microM)-induced death (assessed by nuclear ethidium monoazide bromide staining and anti-tyrosine hydroxylase immunofluorescence microscopy) relative to control preparations. In the experimental group, PC12 cell death was attenuated significantly by the administration of the HO inhibitor, SnMP (1.5 microM), the antioxidant, ascorbate (200 microM), or the iron chelators, deferoxamine (400 microM), and phenanthroline (100 microM). Exposure to conditioned media derived from HO-1 transfected astrocytes also augmented PC12 cell killing in response to dopamine (1 microM) + H(2)O(2) (1 microM) relative to control media. In PD brain, overexpression of HO-1 in nigral astroglia and accompanying iron liberation may facilitate the bioactivation of dopamine to neurotoxic free radical intermediates and predispose nearby neuronal constituents to oxidative damage.