Hepatic ER Stress Research Articles

G-Protein-Coupled Receptors 120 Agonist III Improves Hepatic Inflammation and ER Stress in Steatohepatitis.

GPR120 plays a crucial role in insulin sensitization, inflammatory responses and obesity and is considered as an attractive potential target for the treatment of metabolic dysfunctions. However, the mechanisms of GPR120 agonist III in NAFLD/NASH treatment are still unclear. We aimed to evaluate the effect and molecular mechanisms of GPR120 agonist III on NASH, and search for future treatments of human NAFLD/NASH. The effects of GPR120 agonist III on steatohepatitis were evaluated in mice fed with HFHC diet and MCD diet. The ultrastructural changes of ER were assessed by TEM. Hepatic ROS production was evaluated by DHE staining. Apoptosis and macrophage infiltration were determined by IHC staining. Inflammatory cytokines secretion were examined using mouse XL cytokine array. GPR120 agonist III significantly suppressed macrophage infiltration and ROS production and reversed hepatic inflammation, ER stress and apoptosis in dietary-induced steatohepatitis. GPR120 agonist III will be an attractive treatment method in steatohepatitis, which opens up a new sight for future treatments of human NAFLD/NASH.

Anti-steatotic linagliptin pleiotropic effects encompasses suppression of de novo lipogenesis and ER stress in high-fat-fed mice

AimTo investigate the effects of linagliptin treatment on hepatic energy metabolism and ER stress in high-fat-fed C57BL/6 mice. MethodsForty male C57BL/6 mice, three months of age, received a control diet (C, 10% of lipids as energy, n = 20) or high-fat diet (HF, 50% of lipids as energy, n = 20) for 10 weeks. The groups were randomly subdivided into four groups to receive linagliptin, for five weeks, at a dose of 30 mg/kg/day added to the diets: C, C-L, HF, and HF-L groups. ResultsThe HF group showed higher body mass, total and hepatic cholesterol levels and total and hepatic triacylglycerol levels than the C group, all of which were significantly diminished by linagliptin in the HF-L group. The HF group had higher hepatic steatosis than the C group, whereas linagliptin markedly reduced the hepatic steatosis (less 52%, P < 0.001). The expression of Sirt1 and Pgc1a was more significant in the HF-L group than in the HF group. Linagliptin also elicited enhanced GLP-1 concentrations and a reduction in the expression of the lipogenic genes Fas and Srebp1c. Besides, HF-L showed a reduction in the genes related to endoplasmic reticulum stress Chop, Atf4, and Gadd45 coupled with reduced apoptotic nuclei immunostaining. ConclusionLinagliptin caused a marked reduction in hepatic steatosis as a secondary effect of its glucose-lowering property. NAFLD countering involved reduced lipogenesis, increased beta-oxidation, and relief in endoplasmic reticulum stress, leading to reduced apoptosis and better preservation of the hepatic structure. Therefore, linagliptin may be used, preferably in diabetic patients, to avoid the progression of hepatic steatosis.

Endoplasmic reticulum stress response and pathogenesis of non-alcoholic steatohepatitis

The incidence of chronic liver disease is constantly increasing, owing to the obesity epidemic. Non-alcoholic fatty liver disease (NAFLD) is currently affecting 20-30% of the general population and 75-100% of obese individuals. NAFLD ranges from simple steatosis to damaging non-alcoholic steatohepatitis (NASH), potentially developing into hepatocellular carcinoma. No efficient pharmacological treatment is yet available. During obesity, the hepatic ER stress response can arise from extracellular stress (lipids, glucose, cytokines) and from intracellular stress including lipid buildup in the hepatocyte (steatosis), a hallmark of NAFLD. The chronic activation of the hepatic ER stress response may be a crucial event in the steatosis-NASH transition, triggering cell death, inflammation and accelerating metabolic disorders. We discuss these aspects and we propose that targeting the ER stress response could be effective in treating NAFLD.

Compound Danshen Dripping Pills Prevented Leptin Deficiency-Induced Hepatic ER Stress, Stimulated Autophagy, and Improved Insulin Resistance of ob/ob Mice.

Compound Danshen dripping pills (CDDP) is widely used for the treatment of coronary arteriosclerosis and ischemic heart diseases for decades of years. In our study, we interestingly discovered the effects and mechanism of CDDP on insulin resistance that increase the risk factor of cardiovascular diseases. Effects of CDDP on fasting blood glucose, the insulin tolerance test (ITT), the oral glucose tolerance test (OGTT), hepatic function, and underlying mechanism were analyzed in ob/ob mice. CDDP was found improving the impaired insulin signal sensitivity of ob/ob mice by ameliorating insulin and glucose tolerance, improving hepatic phosphorylation of the insulin receptor substrate-1 on Ser 307 (pIRS1) of ob/ob mice, and restoring hepatic function by decreasing serum ALT and AST, which increased in ob/ob mice serum. Decreasing hepatic phosphorylation of pancreatic ER kinase (PERK) and inositol-requiring enzyme-1 (IRE1) regulating hepatic ER stress in the liver of ob/ob mice were increased by CDDP. Furthermore, CDDP was also found stimulating ob/ob mice hepatic autophagy by increasing the expression of Beclin1 and LC3B, while decreasing P62 expression. Our study discovered an important role of CDDP on improving ob/ob mice insulin resistance and liver function probably through relieving hepatic ER stress and stimulating hepatic autophagy, which would broaden the application value and provide more benefits for treating cardiovascular patients. This trial is registered with NCT01659580.

Phytochemicals in fenugreek seed prevent high fat diet induced metabolic inflammation and NAFLD via the mediation of Akkermansia muciniphila

AbstractIntroductionFenugreek (Trigonella foenum-graecum) is an annual legume that has been used as a spice throughout the world to enhance the sensory quality of foods. A number of health-beneficial bioactive compounds (i.e. trigonelline and diosgenin) have been identified in the seed of fenugreek. These compounds exert multiple health beneficial effects, including anti-obesity and anti-type 2 diabetes. The objective of our study is to determine the underlying mechanism of whole grain of fenugreek seed and purified bioactive compounds, trigonelline and diosgenin, in ameliorating high fat diet (HFD) induced metabolic inflammation and non-alcoholic fatty liver disease (NAFLD).Materials and MethodsTwo groups of C57B6 mice were fed a HFD containing either a vehicle control or 2% of fenugreek seed for 7 weeks. Caloric intake and body weight were monitored weekly during the feeding trial. Glucose tolerance test was conducted to determine the insulin sensitivity. Q-RT-PCR, immunoblotting analysis and histological analysis were used to determine the expression of genes involved in metabolic inflammation and lipid metabolism.Results and DiscussionWhile caloric intake and body weight were comparable between control and fenugreek treated groups during the feeding trial, fenugreek seed containing diet significantly reduced circulation level of inflammatory cytokine TNFa. In the liver, fenugreek suppressed the gene expression of inflammatory cytokines, such as IL-1b and IL-6, and ameliorated hepatic ER stress. The improvement of metabolic inflammation was associated with less activation of genes involved in hepatic de novo lipid synthesis (i.e. FASN and ACC) and enhanced fatty b-oxidation which resulted in significant amelioration of HFD-induced hepatic steatosis, hyperlipidemia and insulin resistance. Analysis the populations of gut microbiota further revealed that the population of Akkermansia muciniphila, a gut bacterial strain that is involved in body weight regulation and insulin sensitivity, was significantly increased by fenugreek seed containing diet. In vitro, treating McA-RH7777, a rat hepatoma cell line, with purified phytochemicals of fenugreek seeds, trigonelline and diosgenin, inhibited hepatic de novo lipogenesis, attenuated ER stress induced by a saturated fatty acid, palmitic acid. These actions improved insulin sensitivity in McA-RH7777 cells by enhancing the functional activity of insulin signaling molecules, such as insulin receptor and AKT.ConclusionOur study unveils a novel mechanism of the bioactive compounds, trigonelline and diosgenin, in fenugreek seed that ameliorate metabolic inflammation and hepatic steatosis via the mediation of gut bacteria. This finding may lend support for developing phytochemicals in fenugreek seed as prebiotic supplement for the prevention and treatment of hyperlipidemia and insulin resistance.

Dapagliflozin not only improves hepatic injury and pancreatic endoplasmic reticulum stress, but also induces hepatic gluconeogenic enzymes expression in obese rats.

With an increasing prevalence of obesity and metabolic syndrome, exploring the effects and delineating the mechanisms of possible therapeutic agents are of critical importance. We examined the effects of SGLT2 inhibitor-dapagliflozin on insulin resistance, hepatic gluconeogenesis, hepatic injury and pancreatic ER stress in high-fat diet-induced obese rats. Male Wistar rats were fed with normal diet (ND) or high-fat diet for 16 weeks. Then high-fat rats were given vehicle (HF) or dapagliflozin (1 mg/kg/day; HFDapa) or metformin (30 mg/kg/day; HFMet) for another 4 weeks. We found that dapagliflozin ameliorated high-fat diet-induced insulin resistance. The fasting plasma glucose level was comparable among groups, although dapagliflozin treatment led to substantial glycosuria. Hepatic gluconeogenic enzymes, PEPCK, G6Pase and FBPase, expression was not different in HF rats compared with ND rats. Meanwhile, dapagliflozin-treated group exhibited the elevation of these enzymes in parallel with the rise of transcription factor CREB, co-factor PGC1α and upstream regulator SIRT1. Hepatic oxidative stress, inflammation and NAFLD activity score as well as hepatic and pancreatic ER stress and apoptosis in obese rats were attenuated by dapagliflozin. We conclude that dapagliflozin improved obesity-related insulin resistance, hepatic and pancreatic injury independent of fasting plasma glucose level. Of note, dapagliflozin-induced glycosuria apparently triggered the up-regulation of hepatic gluconeogenic enzymes to prevent hypoglycemia.

Perinatal protein restriction with postnatal catch-up growth leads to elevated p66Shc and mitochondrial dysfunction in the adult rat liver.

Epidemiological data suggest an inverse relationship between birth weight and long-term metabolic deficits, which is exacerbated by postnatal catch-up growth. We have previously demonstrated that rat offspring subject to maternal protein restriction (MPR) followed by catch-up growth exhibit impaired hepatic function and ER stress. Given that mitochondrial dysfunction is associated with various metabolic pathologies, we hypothesized that altered expression of p66Shc, a gatekeeper of oxidative stress and mitochondrial function, contributes to the hepatic defects observed in MPR offspring. To test this hypothesis, pregnant Wistar rats were fed a control (20% protein) diet or an isocaloric low protein (8%; LP) diet throughout gestation. Offspring born to control dams received a control diet in postnatal life, while MPR offspring remained on a LP diet (LP1) or received a control diet post weaning (LP2) or at birth (LP3). At four months, LP2 offspring exhibited increased protein abundance of both p66Shc and the cis-trans isomerase PIN1. This was further associated with aberrant markers of oxidative stress (i.e. elevated 4-HNE, SOD1 and SOD2, decreased catalase) and aerobic metabolism (i.e., increased phospho-PDH and LDHa, decreased complex II, citrate synthase and TFAM). We further demonstrated that tunicamycin-induced ER stress in HepG2 cells led to increased p66Shc protein abundance, suggesting that ER stress may underlie the programmed effects observed in vivo. In summary, because these defects are exclusive to adult LP2 offspring, it is possible that a low protein diet during perinatal life, a period of liver plasticity, followed by catch-up growth is detrimental to long-term mitochondrial function.

1868-P: Bromocriptine (BC) Improves Hepatic Steatosis, Inflammation, and ER Stress in a Mouse Obesogenic Dietary Model of Nonalcoholic Steatohepatitis (NASH)

The pathogenesis of NASH is a complex interaction among hepatic lipotoxicity, oxidative stress, mitochondrial dysfunction and ER stress leading to hepatic inflammation, cell death, and fibrosis. Bromocriptine, a dopamine D2 receptor agonist is known to reduce adipose lipolysis, plasma hyperFFAemia, and hepatic lipogenesis and inflammation. Also, the antidiabetes drug, bromocriptine-QR has been demonstrated to reduce cardiovascular events. Therefore, we investigated the impact of BC on hepatic pathophysiology in a mouse obesogenic dietary model of NASH. Six-week-old C57B6 male mice (N=8/gr) were maintained on a 40 kcal% fat diet with 5% fructose in the drinking water for 12 weeks to induce NASH then randomized to BC (10mg/kg) or vehicle daily treatment for an additional 3 weeks while maintained on the diet. A group of mice on normal diet was used as a normal reference control. Such BC therapy reduced plasma glucose (28.6%), insulin (31.4%), leptin (42.7%), cholesterol (21.9%) and body weight (14.2%) relative to vehicle (P&amp;lt;0.01). Also, plasma biomarkers of hepatic inflammation: elevated ALT, AST, and LPS, were reduced by BC (32.6, 38.8, and 38.1%, respectively, P&amp;lt;0.05) vs. vehicle. Treatment with BC significantly reduced four gene expression markers each for steatosis (SREBP1c, FASN, ACC1, PLIN2), fatty acid oxidation (PPARα, PGC1α, CPT1, UCP1), inflammation (TNFa, IL1β, MCP1, iNOS), ER stress (DDIT3, ATF, sXBP1, HSPA5), and fibrosis (αSMA, TGFβ, COL1A, TXNIP) by 16-76, 17-47, 31-75, 20-55, and 21-70%, respectively (P&amp;lt;0.05 for each gene). Finally, histological analysis of the liver revealed BC therapy reduced steatosis, inflammation, and ballooning degeneration resulting in a 63% reduced NAFLD activity score (2.6 vs. 7.1, P&amp;lt;0.01). The BC effects on NASH resulted in hepatic physiology closely resembling the normal diet control group. These results suggest that BC could be an effective therapy for NAFLD and NASH. Disclosure T. Tsai: Employee; Self; VeroScience LLC. A. Cincotta: Employee; Self; VeroScience LLC.

APS could potentially activate hepatic insulin signaling in HFD-induced IR mice.

Astragalus polysaccharide (APS) is the main component of Astragalus membranaceus, an anti-diabetic herb being used for thousands of years in Traditional Chinese medicine (TCM). In this study, we aimed to evaluate the impact of APS on hepatic insulin signaling, autophagy and ER stress response in high-fat-diet (HFD)-induced insulin resistance (IR) mice. APS was intra-gastrically administrated and metformin was used as a control medicine. Apart from monitoring the changes in the important parameters of IR progression, the gene and protein expression of the key factors marking the state of hepatic ER stress and autophagic flux were examined. We found that, largely comparable to the metformin regime, APS treatment resulted in an overall improvement of IR, as indicated by better control of body weight and blood glucose/lipid levels, recovery of liver functions and regained insulin sensitivity. In particular, the excessive and pro-apoptotic ER stress response and inhibition of autophagy, as a result of prolonged HFD exposure, were significantly corrected by APS administration, indicating a switch of the cellular fate in favor of cell survival. Using the HepG2/IR cell model, we demonstrated that APS modulated the insulin-initiated phosphorylation cascades in a similar manner to metformin. This study provides a rationale for exploiting the insulin-sensitizing potential of APS, which has a therapeutic performance almost equivalent to metformin, to enrich our options in the treatment of IR.

Hepatic Sdf2l1 controls feeding-induced ER stress and regulates metabolism

Dynamic metabolic changes occur in the liver during the transition between fasting and feeding. Here we show that transient ER stress responses in the liver following feeding terminated by Sdf2l1 are essential for normal glucose and lipid homeostasis. Sdf2l1 regulates ERAD through interaction with a trafficking protein, TMED10. Suppression of Sdf2l1 expression in the liver results in insulin resistance and increases triglyceride content with sustained ER stress. In obese and diabetic mice, Sdf2l1 is downregulated due to decreased levels of nuclear XBP-1s, whereas restoration of Sdf2l1 expression ameliorates glucose intolerance and fatty liver with decreased ER stress. In diabetic patients, insufficient induction of Sdf2l1 correlates with progression of insulin resistance and steatohepatitis. Therefore, failure to build an ER stress response in the liver may be a causal factor in obesity-related diabetes and nonalcoholic steatohepatitis, for which Sdf2l1 could serve as a therapeutic target and sensitive biomarker.

Salvianolic Acid A Attenuates Endoplasmic Reticulum Stress and Protects Against Cholestasis-Induced Liver Fibrosis via the SIRT1/HSF1 Pathway.

Background: Endoplasmic reticulum stress (ER stress) plays a critical role in the pathogenesis of liver fibrosis; thus, it can be a potential therapeutic target of fibrosis. However, the mechanism of ER stress regulation in fibrosis, particularly through sirtuin 1 (SIRT1), remains unclear. The objective of this study was to investigate the effect of SIRT1-mediated inhibition of ER stress in bile duct ligation (BDL)-induced liver fibrosis, and to explore the effect of salvianolic acid A (SalA) on BDL-induced liver fibrosis through SIRT1/heat shock factor 1 (HSF1) signaling.Materials and Methods: We explored the effects of SalA on liver fibrosis and ER stress in BDL-induced liver fibrosis in rats and the human hepatic stellate cell line LX2 cells. The LX2 cells were treated with 20 ng of platelet-derived growth factor-BB homodimer (PDGF-BB) for 24 h, and then incubated in the absence or presence of SalA (25 μM) for 24 h.Results: In vivo, SalA treatment alleviated BDL-induced liver injury and ER stress. Importantly, SalA treatment increased HSF1 expression and activity using a SIRT1-dependent mechanism. In LX2 cells, PDGF-BB induced ER stress and fibrosis were blocked by HSF1 overexpression. Furthermore, SIRT1 siRNA abrogated the SalA-mediated promotion of HSF1 deacetylation and expression, suggesting that SalA-mediated protection occurs by SIRT1 targeting HSF1 for deacetylation.Conclusion: This is the first study to identify the SIRT1/HSF1 pathway as a key therapeutic target for controlling BDL-induced liver fibrosis and to show that SalA confers protection against BDL- and PDGF-BB-induced hepatic fibrosis and ER stress through SIRT1-mediated HSF1 deacetylation.

Gamma-Tocotrienol Attenuates the Hepatic Inflammation and Fibrosis by Suppressing Endoplasmic Reticulum Stress in Mice.

Gamma-tocotrienol (γT3), an unsaturated isoform of vitamin E, is implicated in the hepatoprotective effects. The aim is to determine the effectiveness of γT3 on nonalcoholic fatty liver disease (NAFLD). C57BL/6 male mice are fed a diet containing high fat (45%) and cholesterol (0.2%) along with sucrose drink (HFCS) or HFCS diet supplemented with 0.1% γT3 (HFCS+γT3). The inclusion of γT3 robustly decreases the HFCS diet-induced de novo lipogenesis (DNL), ER stress, and inflammation leading to reduced hepatic steatosis and fibrosis. Next, mice are fed a methionine- and choline-deficient (MCD) diet or MCD diet with γT3 (MCD+γT3). The γT3 supplementation significantly reduces the MCD diet-induced hepatic ER stress and fibrosis despite the minimal impact on steatosis. To further investigate the role of ER stress, the mice with genetic ablation of CHOP are fed an MCD or MCD+γT3 diet. CHOP deletion abolishes the γT3-mediated suppression of hepatic fibrosis, suggesting that modulation of ER stress is a prerequisite to inhibit hepatic inflammation and fibrosis. γT3 supplementation is effective in attenuating NAFLD and fibrosis through a synergistic mechanism of decreased DNL and hepatic ER stress. This work strongly supports the translational potential of γT3 supplementation against NAFLD.

ILDR2 Alters Hepatic Phospholipid Composition via MBOAT7—Effects on Liver Fat

Nonalcoholic fatty liver disease (NAFLD) is associated with obesity and type 2 diabetes, and increases the risk for liver cirrhosis and cancer. A diabetes susceptibility gene, ILDR2, encodes a transmembrane protein localized to the ER membrane whose primary physiological functions are unclear. We previously purified ILDR2-containing protein complexes using the tandem affinity purification tags and identified MBOAT7, which mediates acyl-chain remodeling of phosphatidylinositols (PIs). The MBOAT7 variant rs641738 T allele is associated with decreased hepatic PI (36:4) and PI (38:3) and an increase in severity of NAFLD. We hypothesized that ILDR2 may influence hepatic Pl composition in NAFLD. We used two mouse models fed a methionine- and choline-deficient (MCD) diet: 1. liver-specific ILDR2 knockout (LKO-ILDR2) using an AAV-Cre/loxP system; and 2. AAV-overexpressing ILDR2 (OX-ILDR2) mice. In LKO-ILDR2 mice compared to AAV-GFP injected ILDR2 floxed (GFP-flox) mice, liver triglyceride (TG) content was significantly increased by 1.6-fold with a significant 1.2-fold increase in plasma TG. In LKO-ILDR2 mice compared to GFP-flox mice, hepatic PI (38:4) was significantly decreased. The livers of LKO-ILDR2 mice showed significantly increased JNK activation, collagen gene expression, and increased in hydroxyproline, suggesting that ILDR2 depletion leads to inflammation and the development of fibrosis. In OX-ILDR2 mice compared to AAV-GFP injected WT (GFP-WT) mice, hepatic PI and PC species were significantly increased. The liver in OX-ILDR2 mice showed decreased expression of JNK and ER stress-related genes, and hydroxyproline, with a slight (9%) decrease in hepatic TG compared to GFP-WT mice. Thus, ILDR2 overexpression attenuated hepatic inflammation, ER stress, and fibrogenesis. Manipulation of hepatic ILDR2 expression is associated with changes in PI and PC molecular species in the liver. Patterns of hepatic phosphatidyl fatty acid composition are associated with NAFLD development. Disclosure K. Watanabe: None. K. Nakayama: None. K. Yoshida: None. E.J. Millings: None. C.A. LeDuc: None. R. Leibel: None. S. Iwamoto: None.

988 - Aldose Reductase Null Mice are Protected Against Alcohol-Induced Hepatic ER Stress, Apoptosis and Liver Injury

988 - Aldose Reductase Null Mice are Protected Against Alcohol-Induced Hepatic ER Stress, Apoptosis and Liver Injury

Adipose-specific lipin1 overexpression in mice protects against alcohol-induced liver injury

Excessive fatty acid release from the white adipose tissue (WAT) contributes to the development of alcoholic liver disease (ALD). Lipin1 (LPIN1), as a co-regulator of DNA-bound transcription factors and a phosphatidic acid (PA) phosphatase (PAP) enzyme that dephosphorylates PA to form diacylglycerol (DAG), is dramatically reduced by alcohol in the WAT. This study aimed at determining the role of adipose LPIN1 in alcohol-induced lipodystrophy and the development of ALD. Transgenic mice overexpressing LPIN1 in adipose tissue (LPIN1-Tg) and wild type (WT) mice were fed a Lieber-DeCarli alcohol or isocaloric maltose dextrin control liquid diet for 8 weeks. Alcohol feeding to WT mice resulted in significant liver damage, which was significantly alleviated in the LPIN1-Tg mice. Alcohol feeding significantly reduced epididymal WAT (EWAT) mass, inhibited lipogenesis, and increased lipolysis in WT mice, which were attenuated in the LPIN1-Tg mice. LPIN1 overexpression also partially reversed alcohol-reduced plasma leptin levels. In WT mice, alcohol feeding induced hepatic lipid accumulation and down-regulation of beta-oxidation genes, which were dramatically alleviated in the LPIN1-Tg mice. LPIN1 overexpression also significantly attenuated alcohol-induced hepatic ER stress. These results suggest that overexpression of LPIN1 in adipose tissue restores WAT lipid storage function and secretive function to alleviate alcohol-induced liver injury.

Vildagliptin Can Alleviate Endoplasmic Reticulum Stress in the Liver Induced by a High Fat Diet.

Purpose. We investigated whether a DDP-4 inhibitor, vildagliptin, alleviated ER stress induced by a high fat diet and improved hepatic lipid deposition. Methods. C57BL/6 mice received standard chow diet (CD), high fat diet (HFD), and HFD administered with vildagliptin (50 mg/Kg) (V-HFD). After administration for 12 weeks, serum alanine aminotransferase, glucose, cholesterol, triglyceride, and insulin levels were analyzed. Samples of liver underwent histological examination and transmission electron microscopy, real-time PCR for gene expression levels, and western blots for protein expression levels. ER stress was induced in HepG2 cells with palmitic acid and the effects of vildagliptin were investigated. Results. HFD mice showed increased liver weight/body weight (20.27%) and liver triglycerides (314.75%) compared to CD mice, but these decreased by 9.27% and 21.83%, respectively, in V-HFD mice. In the liver, HFD induced the expression of ER stress indicators significantly, which were obviously decreased by vildagliptin. In vitro, the expressions of molecular indicators of ER stress were reduced in HepG2 when vildagliptin was administered. Conclusions. Vildagliptin alleviates hepatic ER stress in a mouse high fat diet model. In HepG2 cells, vildagliptin directly reduced ER stress. Therefore, vildagliptin may be a potential agent for nonalcoholic fatty liver disease.

Mitochondrial DNA-enriched microparticles promote acute-on-chronic alcoholic neutrophilia and hepatotoxicity.

Over the last several years, one of the major advances in the field of alcoholic liver disease research was the discovery that binge alcohol consumption induced neutrophilia and hepatic neutrophil infiltration in chronically ethanol-fed mice and human subjects with excessive alcohol use (EAU); however, the underlying mechanisms remain obscure. Here, we demonstrated that chronic EAU patients with a history of recent excessive drinking (EAU + RD) had higher serum levels of mitochondrial DNA (mtDNA)-enriched microparticles (MPs) than EAU without recent drinking (EAU - RD) and healthy controls, which correlated positively with circulating neutrophils. Similarly, mice with chronic-plus-binge (E10d + 1B) ethanol feeding also had markedly elevated serum levels of mtDNA-enriched MPs, with activation of hepatic ER stress and inflammatory responses. Inhibition of ER stress by gene KO or inhibitors attenuated ethanol-induced elevation of mtDNA-enriched MPs, neutrophilia, and liver injury. The data from the study of hepatocyte-specific deletion of the protein kinase RNA-like ER kinase (Perk) gene in mice and of cultured hepatocytes demonstrated that hepatocytes were the main source of mtDNA-enriched MPs after ethanol feeding. Finally, administration of mtDNA-enriched MPs isolated from E10d+1B-fed mice caused neutrophilia in mice. In conclusion, E10d + 1B ethanol consumption activates hepatic ER stress-dependent mtDNA-enriched MP release, leading to neutrophilia and liver injury.

Polytrauma-induced hepatic stress response and the development of liver insulin resistance

Insulin resistance and metabolic dysfunction are common following injury. Polytrauma is defined as combined injuries to more than one body part or organ system, and is common in modern warfare, as well as automobile and industrial accidents. Polytrauma can include any combination of burn injury, fracture, hemorrhage, trauma to the extremities, and blunt or penetrating trauma. Multiple minor injuries are often more deleterious than a more severe single injury. To investigate the mechanisms of development of insulin resistance following injury, we have developed a rat model of polytrauma which combined soft tissue trauma with burn injury and penetrating gastrointestinal (GI) trauma. Male Sprague-Dawley rats were subjected to a laparotomy plus either a 15–18% total body surface area scald burn or a single puncture of the cecum (CLP) with a G30 needle, or the combination of both burn and CLP injuries (polytrauma). We examined the effects of polytrauma which increased markers of hepatic endoplasmic reticulum (ER) stress, and increased hepatic Trib3 mRNA levels coincident with reduced insulin-inducible insulin signaling. Phosphorylation/activation of the insulin receptor (IR) and AKT were decreased at 24, but not 6h following polytrauma. These results demonstrate a complex, time-dependent development of hepatic ER-stress and a diminished response to insulin, which were among the pathological sequelae following polytrauma.

High‐sucrose Diet Time‐dependently Changes Hepatic Endoplasmic Reticulum Stress from an Adaptive to Pro‐apoptotic Pattern in Young Mice

The relationship between excessive nutrient intake and hepatic endoplasmic reticulum stress (ER stress) is well stablished. However, mechanisms underlying the progression from adaptive to apoptotic pattern are still unclear. This study sought to investigate the effects of high‐sucrose diet on metabolic disorders associated to hepatic ER stress. Weaned Swiss mice received standard chow (CTR) or high‐sucrose diet (HSD; 25% sucrose) for distinct periods of 30 (CTR30 and HSD30) and 60 (CTR60 and HSD60) days. Weight gain, food intake, feed efficiency and Lee index (LI) were measured for obesity development assessment. At each treatment end, glucose (GTT) and insulin (ITT) tolerance tests were performed. Blood, white adipose tissue (WAT) depots and liver were collected upon euthanasia of anesthetized animals. Serum glucose (GL), triglycerides (TG), total cholesterol (TC) and free fatty acids (FFA) levels were determined. WAT depots were weighed and their lipolytic activity assessed. Hepatic tissue was used to determine the lipid profile and gene expression of ER stress (ATF6, IRE1α, PERK, GRP78, PDI, NRF2) and pro‐apoptotic (CHOP and caspase 2) markers. Only HSD60 showed higher weight gain (8%), LI (3%) and feed efficiency (31%), as compared to CTR60. WAT was 2‐fold higher in HSD60 than in HSD30, whereas both of them were significantly higher than their controls. Lipolytic activity under isoproterenol‐stimulation was 40% lower in HSD60. Serum GL and TC were increased in 20 and 40%, respectively, in both HSD groups. However, TG (43%) and FFA (25%) were increased only in HSD60. Area under the curve of GTT was augmented in both HSD30 (14%) and HSD60 (41%), as compared to their controls. Whereas only HSD60 presented reduced kITT (37%) and increase TyG (8%). Total fat liver content was increased in 22 and 61% for HSD30 and HSD60, respectively. On the other hand, hepatic TG was augmented only in HSD60 (69%). HSD30 presented increased hepatic gene expression of GRP78 (57%), PERK (83%), IRE1α (49%), ATF6 (70%), PDI (41%) and NRF2 (32%), and reduced expression of CHOP (39%). An opposite pattern was found in HSD60, with reduced expression of PERK (34%), IRE1α (52%), ATF6 (36%) and PDI (46%), and increase expression of CHOP (72%). Notwithstanding, caspase‐2 expression did not change in any group. Therefore, our data suggest the HSD intake for up to 60 days after weaning rapidly promotes metabolic disorders associate to early onset of hepatic ER stress, which promptly progress from an adaptive to a pro‐apoptotic pattern.Support or Funding InformationFAPEMA, CNPq, CAPES and UFMA.

Hepatic rhythmicity of endoplasmic reticulum stress is disrupted in perinatal and adult mice models of high-fat diet-induced obesity

We investigated the regulation of hepatic ER stress in healthy liver and adult or perinatally programmed diet-induced non-alcoholic fatty liver disease (NAFLD). Female mice were fed either obesogenic or control diet before mating, during pregnancy and lactation. Post-weaning, offspring from each maternal group were divided into either obesogenic or control diet. At six months, offspring were sacrificed at 4-h intervals over 24 h. Offspring fed obesogenic diets developed NAFLD phenotype, and the combination of maternal and offspring obesogenic diets exacerbated this phenotype. UPR signalling pathways (IREα, PERK, ATF6) and their downstream regulators showed different basal rhythmicity, which was modified in offspring exposed to obesogenic diet and maternal programming. The double obesogenic hit increased liver apoptosis measured by TUNEL staining, active caspase-3 and phospho-JNK and GRP78 promoter methylation levels. This study demonstrates that hepatic UPR is rhythmically activated. The combination of maternal obesity (MO) and obesogenic diets in offspring triggered altered UPR rhythmicity, DNA methylation and cellular apoptosis.