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Hepatic ERK2 Deficiency Develops Hepatic ER Stress with Malfunction of Metabolism, Which Causes Endothelial Dysfunction
'LDEHWHV0HWDEROLF6\QGURPH 23 +HSDWLF(5.'HILFLHQF\'HYHORSV+HSDWLF(56WUHVV ZLWK0DOIXQFWLRQRI0HWDEROLVP:KLFK&DXVHV (QGRWKHOLDO'\VIXQFWLRQ
Takeshi Adachi1, Toyokazu Kimura1, Atsushi Sato1, Takayuki Namba1, Shunpei Horii1, Hirotaka Yada1, Takehiko Kujiraoka1, and Yasushi Sato1 1 National Defense Medical College, Japan We previously found that liver-specific extracellular regulatory kinase2 (ERK2) knockout mice (LE2KO) with high-fat/highsucrose diet (HFHSD) revealed severe hepatosteatosis (HST) and endothelial dysfunction. The aim of the study is to elucidate the mechanisms to impair nitric oxide (NO)/reactive oxygen species (ROS) balance with HST in LE2KO with HFHSD for 20 weeks. The hepatic endoplasmic reticulum (ER) stress with the expression of hepatic ER Ca2+-ATPase-2 (SERCA2) were evaluated. Vascular sROS were assessed with dihydroethidium staining and Amplex red assay with the expressions of NADPH oxidase isoforms (NOX). The endothelial function with the endothelial NO synthase (eNOS) phosphorylation was tested. LE2KO fed with HFHSD revealed the exacerbation of HST, the induction of hepatic ER stress with CHOP/BiP expressions, the decreased SERCA2 expression, and the induction of hepatic NOX2. . LE2KO with HFHSD increased the serum fasting/fed glucose, free fatty acids, homocysteine, asymmetric dimethylarginine, ornithine/arginine ratio, interleukin (IL)-1Į, and leptin. The serum adiponectin levels were decreased with enlarged adipocytes size. Vascular ROS were elevated with upregulation of NOX1/4 and the decreased phosphorylation of eNOS at ser1177 with the remarkable endothelial dysfunction were observed in LE2KO with HFHSD. Conclusion: LE2KO with HFHSD induced ER stress with the decreased SERCA expression, resulted in HST. The induction of HST was associated with malfunction of hepatic metabolism and the elevated serum insulin, glucose, and FFA, the altered NOrelated amino acids metabolites, and the dysfunctional release of adipocytokines with adipocytes enlargement. These factors caused NO/ROS imbalance and induced endothelial dysfunction.
doi: xxxxx doi: 10.1016/j.freeradbiomed.2015.10.060 24 $UH3DQFUHDWLFȕ&HOOV5HDOO\6HQVLWLYHWR5HDFWLYH 6SHFLHV" Katarzyna Broniowska1, Bryndon Oleson1, and John Corbett1 1 Medical College of Wisconsin, USA
In both type 1 and 2 diabetes oxidative stress is believed to contribute to dysfunction and death of insulin-VHFUHWLQJ ȕ-cells. 3DQFUHDWLFȕ-cells are typically regarded as particularly vulnerable to oxidant-induced damage due to low levels of expression of antioxidant defense enzymes (e.g., catalase). Here, we determined the effects or reactive oxygen and nitrogen species on IXQFWLRQ DQG YLDELOLW\ RI ȕ-FHOOV 7UHDWPHQW RI ȕ-cell line INS 832/13 with either nitric oxide, superoxide, or H 2O2 results in LPSDLUHG PLWRFKRQGULDO R[LGDWLRQ DQG FDXVHV ȕ-cell death. In contrast, peroxynitrite does not impair oxidative metabolism or GHFUHDVH ȕ-cell viability. When exposed to H2O2 generated by glucose oxidase to provide a continuous release of H 2O2, we show WKDW SDQFUHDWLF ȕ-cells are capable of metabolizing H2O2.
S22
The detoxification of H2O2 occurs at concentrations of H2O2 under a threshold of ~ 100 PM. At concentrations at or above the threshold, poly-ADP ribose polymerase (PARP)-dependent depletion of NAD+ and ATP is responsible for the decrease in cell viability. It is only possible to measure H2O2 PHWDEROLVPE\ȕ-cell under conditions of continues release. In response to a bolus addition, H2O2 concentrations decrease within minutes and the loss of H2O2 is independent of cell presence. 7KHVH ILQGLQJV GHPRQVWUDWH WKDW ȕ-cells, despite the notion of being ill-equipped to detoxify oxidants, are resistant to peroxynitrite and have the capacity to metabolize H2O2. This study also highlights the importance of using continuous delivery of oxidants rather than bolus addition.
doi: xxxxx doi: 10.1016/j.freeradbiomed.2015.10.061 25 3XOPRQDU\0\HORSHUR[LGDVH$FWLYLW\DQG,QVXOLQ 5HVLVWDQFHLQ2EHVLW\
Maria Cecilla Della Vedova1, Marcos D Munoz1, Silvina Garcia1, Sandra E Gomez Mejiba1, and Dario C Ramirez1 1 CONICET, UNSL, San Luis, Argentina In obesity, inflamed adipose tissue releases into circulation a number of mediators causing insulin resistance (IR) and other metabolic abnormalities. These mediators are involved in neutrophilic inflammation (NI) in a number of tissues. Activated neutrophils contain myeloperoxidase (MPO) that produce HOCl. Clorotyrosine is a marker of HOCl protein oxidation. Herein we used a diet-induced obesity (DIO) model that resembles many of the features of metabolic syndrome to evaluate NI in the lung and how it affects peripheral IR. B6 mice were fed for 16 weeks with either a high-fat diet (HFD, 22% chicken fat) and 10% fructose in the drinking water (obese mice) or a low-fat diet, 6% chicken fat) and tap water (lean mice). Compared to lean, the obese lung had more neutrophils, MPO and markers of protein oxidation (chlorotyrosine and carbonyls) and reduced antioxidant capacity (enzyme activity, GSH, ascorbate). In relation to lean, obese animals had more inflammatory and oxidative stress markers in serum; and had more IR. When these animals were instilled daily for 7 days before the sacrifice with either 2.5 nmol of DMPO²to reduce retention of neutrophils in the lung, 1 nmol of ABAH²to inhibit HOCl production by MPO, 5 nmol taurine or 1 nmol resveratrol²to scavenge HOCl; we observed reduced lung and systemic MPO activity, clorotyrosine, oxidative stress/ inflammation and peripheral IR compared to non-treated obese animals. Conversely, instillation of lean and obese mice with 50Pg lipopolisaccharide has increased pulmonary MPO, chlorotyrosine, systemic inflammation and were more IR. These data are consistent with a critical role of neutrophils and MPO-derived HOCl in worsening IR and systemic inflammation in obese subjects, eVSHFLDOO\WKRVHH[SRVHGWRDLUZD\V¶LUULWDQWV PICT-2014-3369 (to DCR), PROICO-2-3214 (To DCR) and PROICO10-0414(To SEGM).
doi: xxxxx doi: 10.1016/j.freeradbiomed.2015.10.062
SFRBM 2015
Takeshi Adachi1, Toyokazu Kimura1, Atsushi Sato1, Takayuki Namba1, Shunpei Horii1, Hirotaka Yada1, Takehiko Kujiraoka1, and Yasushi Sato1 1 National Defense Medical College, Japan We previously found that liver-specific extracellular regulatory kinase2 (ERK2) knockout mice (LE2KO) with high-fat/highsucrose diet (HFHSD) revealed severe hepatosteatosis (HST) and endothelial dysfunction. The aim of the study is to elucidate the mechanisms to impair nitric oxide (NO)/reactive oxygen species (ROS) balance with HST in LE2KO with HFHSD for 20 weeks. The hepatic endoplasmic reticulum (ER) stress with the expression of hepatic ER Ca2+-ATPase-2 (SERCA2) were evaluated. Vascular sROS were assessed with dihydroethidium staining and Amplex red assay with the expressions of NADPH oxidase isoforms (NOX). The endothelial function with the endothelial NO synthase (eNOS) phosphorylation was tested. LE2KO fed with HFHSD revealed the exacerbation of HST, the induction of hepatic ER stress with CHOP/BiP expressions, the decreased SERCA2 expression, and the induction of hepatic NOX2. . LE2KO with HFHSD increased the serum fasting/fed glucose, free fatty acids, homocysteine, asymmetric dimethylarginine, ornithine/arginine ratio, interleukin (IL)-1Į, and leptin. The serum adiponectin levels were decreased with enlarged adipocytes size. Vascular ROS were elevated with upregulation of NOX1/4 and the decreased phosphorylation of eNOS at ser1177 with the remarkable endothelial dysfunction were observed in LE2KO with HFHSD. Conclusion: LE2KO with HFHSD induced ER stress with the decreased SERCA expression, resulted in HST. The induction of HST was associated with malfunction of hepatic metabolism and the elevated serum insulin, glucose, and FFA, the altered NOrelated amino acids metabolites, and the dysfunctional release of adipocytokines with adipocytes enlargement. These factors caused NO/ROS imbalance and induced endothelial dysfunction.
doi: xxxxx doi: 10.1016/j.freeradbiomed.2015.10.060 24 $UH3DQFUHDWLFȕ&HOOV5HDOO\6HQVLWLYHWR5HDFWLYH 6SHFLHV" Katarzyna Broniowska1, Bryndon Oleson1, and John Corbett1 1 Medical College of Wisconsin, USA
In both type 1 and 2 diabetes oxidative stress is believed to contribute to dysfunction and death of insulin-VHFUHWLQJ ȕ-cells. 3DQFUHDWLFȕ-cells are typically regarded as particularly vulnerable to oxidant-induced damage due to low levels of expression of antioxidant defense enzymes (e.g., catalase). Here, we determined the effects or reactive oxygen and nitrogen species on IXQFWLRQ DQG YLDELOLW\ RI ȕ-FHOOV 7UHDWPHQW RI ȕ-cell line INS 832/13 with either nitric oxide, superoxide, or H 2O2 results in LPSDLUHG PLWRFKRQGULDO R[LGDWLRQ DQG FDXVHV ȕ-cell death. In contrast, peroxynitrite does not impair oxidative metabolism or GHFUHDVH ȕ-cell viability. When exposed to H2O2 generated by glucose oxidase to provide a continuous release of H 2O2, we show WKDW SDQFUHDWLF ȕ-cells are capable of metabolizing H2O2.
S22
The detoxification of H2O2 occurs at concentrations of H2O2 under a threshold of ~ 100 PM. At concentrations at or above the threshold, poly-ADP ribose polymerase (PARP)-dependent depletion of NAD+ and ATP is responsible for the decrease in cell viability. It is only possible to measure H2O2 PHWDEROLVPE\ȕ-cell under conditions of continues release. In response to a bolus addition, H2O2 concentrations decrease within minutes and the loss of H2O2 is independent of cell presence. 7KHVH ILQGLQJV GHPRQVWUDWH WKDW ȕ-cells, despite the notion of being ill-equipped to detoxify oxidants, are resistant to peroxynitrite and have the capacity to metabolize H2O2. This study also highlights the importance of using continuous delivery of oxidants rather than bolus addition.
doi: xxxxx doi: 10.1016/j.freeradbiomed.2015.10.061 25 3XOPRQDU\0\HORSHUR[LGDVH$FWLYLW\DQG,QVXOLQ 5HVLVWDQFHLQ2EHVLW\
Maria Cecilla Della Vedova1, Marcos D Munoz1, Silvina Garcia1, Sandra E Gomez Mejiba1, and Dario C Ramirez1 1 CONICET, UNSL, San Luis, Argentina In obesity, inflamed adipose tissue releases into circulation a number of mediators causing insulin resistance (IR) and other metabolic abnormalities. These mediators are involved in neutrophilic inflammation (NI) in a number of tissues. Activated neutrophils contain myeloperoxidase (MPO) that produce HOCl. Clorotyrosine is a marker of HOCl protein oxidation. Herein we used a diet-induced obesity (DIO) model that resembles many of the features of metabolic syndrome to evaluate NI in the lung and how it affects peripheral IR. B6 mice were fed for 16 weeks with either a high-fat diet (HFD, 22% chicken fat) and 10% fructose in the drinking water (obese mice) or a low-fat diet, 6% chicken fat) and tap water (lean mice). Compared to lean, the obese lung had more neutrophils, MPO and markers of protein oxidation (chlorotyrosine and carbonyls) and reduced antioxidant capacity (enzyme activity, GSH, ascorbate). In relation to lean, obese animals had more inflammatory and oxidative stress markers in serum; and had more IR. When these animals were instilled daily for 7 days before the sacrifice with either 2.5 nmol of DMPO²to reduce retention of neutrophils in the lung, 1 nmol of ABAH²to inhibit HOCl production by MPO, 5 nmol taurine or 1 nmol resveratrol²to scavenge HOCl; we observed reduced lung and systemic MPO activity, clorotyrosine, oxidative stress/ inflammation and peripheral IR compared to non-treated obese animals. Conversely, instillation of lean and obese mice with 50Pg lipopolisaccharide has increased pulmonary MPO, chlorotyrosine, systemic inflammation and were more IR. These data are consistent with a critical role of neutrophils and MPO-derived HOCl in worsening IR and systemic inflammation in obese subjects, eVSHFLDOO\WKRVHH[SRVHGWRDLUZD\V¶LUULWDQWV PICT-2014-3369 (to DCR), PROICO-2-3214 (To DCR) and PROICO10-0414(To SEGM).
doi: xxxxx doi: 10.1016/j.freeradbiomed.2015.10.062
SFRBM 2015