Nanoformulated SOD1 ameliorates the combined NASH and alcohol-associated liver disease partly via regulating CYP2E1 expression in adipose tissue and liver.

Enhanced free fatty acid (FFA) flux from adipose tissue (AT) to liver plays an important role in the development of nonalcoholic steatohepatitis (NASH) and alcohol-associated liver disease (AALD). We determined the effectiveness of nanoformulated superoxide dismutase 1 (Nano) in attenuating liver injury in a mouse model exhibiting a combination of NASH and AALD. Male C57BL6/J mice were fed a chow diet (CD) or a high-fat diet (HF) for 10 wk followed by pair feeding of the Lieber-DeCarli control (control) or ethanol (ET) diet for 4 wk. Nano was administered once every other day for the last 2 wk of ET feeding. Mice were divided into 1) CD + control diet (CD + Cont), 2) high-fat diet (HF) + control diet (HF + Cont), 3) HF + Cont + Nano, 4) HF + ET diet (HF + ET), and 5) HF + ET + Nano. The total fat mass, visceral AT mass (VAT), and VAT perilipin 1 content were significantly lower only in HF + ET-fed mice but not in HF + ET + Nano-treated mice compared with controls. The HF + ET-fed mice showed an upregulation of VAT CYP2E1 protein, and Nano abrogated this effect. We noted a significant rise in plasma FFAs, ALT, and monocyte chemoattractant protein-1 in HF + ET-fed mice, which was blunted in HF + ET + Nano-treated mice. HF + ET-induced increases in hepatic steatosis and inflammatory markers were attenuated upon Nano treatment. Nano reduced hepatic CYP2E1 and enhanced catalase levels in HF + ET-fed mice with a concomitant increase in SOD1 protein and activity in liver. Nano was effective in attenuating AT and liver injury in mice exhibiting a combination of NASH and AALD, partly via reduced CYP2E1-mediated ET metabolism in these organs.NEW & NOTEWORTHY Increased free fatty acid flux from adipose tissue (AT) to liver accompanied by oxidative stress promotes nonalcoholic steatohepatitis (NASH) and alcohol-associated liver injury (AALD). Obesity increases the severity of AALD. Using a two-hit model involving a high-fat diet and chronic ethanol feeding to mice, and treating them with nanoformulated superoxide dismutase (nanoSOD), we have shown that nanoSOD improves AT lipid storage, reduces CYP2E1 in AT and liver, and attenuates the combined NASH/AALD in mice.

Effect of nanoformulated copper/zinc superoxide dismutase on chronic ethanol-induced alterations in liver and adipose tissue.

BACKGROUND: We previously reported that nanoformulated copper/zinc superoxide dismutase (Nano) attenuates non-alcoholic fatty liver disease and adipose tissue (AT) inflammation in obese animals. Here, we sought to determine whether Nano treatment attenuates alcohol-associated liver disease (AALD) and AT inflammation in alcohol-fed mice. METHODS: We pre-treated E-47 cells (HepG2 cells that over-express CYP2E1) with native- or nano-superoxide dismutase (SOD) for 6 h, followed by treatment with ethanol and/or linoleic acid (LA), a free fatty acid. For in vivo studies, male C57BL/6 mice were fed the Lieber-DeCarli control or ethanol liquid diet for 4 weeks. The mice received Nano once every 2 days during the last 2 weeks of ethanol feeding. RESULTS: Our in vitro studies revealed that Nano pretreatment reduced LA + ethanol-induced oxidative stress in E-47 cells. Our in vivo experiments showed that ethanol-fed Nano-treated mice had 22% lower hepatic triglyceride levels than mice fed ethanol alone. Nano-treated ethanol-fed mice also had 2-fold lower levels of Cd68 and similarly reduced levels of Ccl2 and Mmp12 mRNAs, than in untreated ethanol-fed mice. We also noted that ethanol feeding caused a remarkable increase in hepatic and/or plasma MCP-1 and CCR2 protein, which was blunted in ethanol + Nano-treated animals. The hepatic content of SREBP-1c, a transcription factor that promotes lipogenesis, was higher in ethanol-fed mice than controls but was attenuated in ethanol + Nano-treated animals. Further, livers of ethanol + Nano-treated mice had significantly higher levels of phosphorylated adenosine monophosphate-activated protein kinase (AMPK) than both control and ethanol-fed mice. In AT, the levels of Il6 mRNA, a hepatoprotective cytokine, and that of Arg1, a marker of anti-inflammatory macrophages, were significantly increased in ethanol + Nano-treated mice compared with control mice. CONCLUSION: Our data indicate that Nano treatment attenuates ethanol-induced steatohepatitis and that this effect is associated with an apparent activation of AMPK signaling. Our data also suggest that Nano induces Arg1 and Il6 expression in AT, suggesting anti-inflammatory effects in this tissue.

Nanoformulated copper/zinc superoxide dismutase exerts differential effects on glucose vs lipid homeostasis depending on the diet composition possibly via altered AMPK signaling.

Evidence suggests that superoxide dismutase 1 (SOD1) promotes glucose vs lipid metabolism depending on the diet type. We recently reported that nanoformulated SOD1 (Nano) improved lipid metabolism without altering glucose homeostasis in high-fat (HF) diet-fed mice. Here, we sought to determine the effects and potential mechanisms of Nano in modulating glucose and lipid homeostasis in mice fed a normal chow diet (CD) vs HF diet. Mice were fed a CD or a HF diet (45%) for 10 wk and injected with Nano once every 2 days for 15 days. The fasting glucose level was lower (P < 0.05) in CD + Nano-treated mice compared to control. Conversely, blood glucose was not altered but serum triglycerides were lower in HF + Nano-treated mice. Genes involved in fatty acid synthesis were reduced by Nano in the skeletal muscle of CD but not of HF diet-fed mice. Adenosine monophosphate-activated protein kinase (AMPK), which promotes both glucose and lipid metabolism depending on the fuel availability, is activated by Nano in CD-fed mice. Moreover, Nano increased phosphorylation of ACC, a downstream target of AMPK, in both CD and HF diet-fed mice. Nano increased mitochondrial respiration in C2C12 myocytes in the presence of glucose or fatty acid, and this effect is inhibited by Compound C, an AMPK inhibitor. Our data suggest that Nano promotes glucose and lipid metabolism in CD and HF diet-fed mice, respectively, and this effect is mediated partly via AMPK signaling.

A combination of dietary N-3 fatty acids and a cyclooxygenase-1 inhibitor attenuates nonalcoholic fatty liver disease in mice.

We sought to determine whether a combination of purified n-3 fatty acids (n-3) and SC-560 (SC), a cyclooxygenase-1-specific inhibitor, is effective in ameliorating nonalcoholic fatty liver disease in obesity. Female wild-type mice were fed a high-fat and high-cholesterol diet (HF) supplemented with n-3 in the presence or absence of SC. Mice treated with SC alone exhibited no change in liver lipids, whereas n-3-fed mice tended to have lower hepatic lipids. Mice given n-3+SC had significantly lower liver lipids compared with HF controls indicating enhanced lipid clearance. Total and sulfated bile acids were significantly higher only in n-3+SC-treated mice compared with chow diet (CD) controls. Regarding mechanisms, the level of pregnane X receptor (PXR), a nuclear receptor regulating drug/bile detoxification, was significantly higher in mice given n-3 or n-3+SC. Studies in precision-cut liver slices and in cultured hepatoma cells showed that n-3+SC enhanced not only the expression/activation of PXR and its target genes but also the expression of farnesoid X receptor (FXR), another regulator of bile synthesis/clearance, indicating that n-3+SC can induce both PXR and FXR. The mRNA level of FGFR4 which inhibits bile formation showed a significant reduction in Huh 7 cells upon n-3 and n-3+SC treatment. PXR overexpression in hepatoma cells confirmed that n-3 or SC each induced the expression of PXR target genes and in combination had an enhanced effect. Our findings suggest that combining SC with n-3 potentiates its lipid-lowering effect, in part, by enhanced PXR and/or altered FXR/FGFR4 signaling.

Nanoformulated copper/zinc superoxide dismutase reduces adipose inflammation in obesity.

OBJECTIVE: An intimate association exists between oxidative stress and inflammation. Because adipose tissue (AT) inflammation is intricately linked to metabolic disorders, it was hypothesized that reducing oxidative stress would be effective in ameliorating AT inflammation in obesity. METHODS: Wild-type mice were fed a high-fat diet (HF) for 8 weeks followed by a 2-week treatment with nanoformulated copper/zinc superoxide dismutase (NanoSOD). The mice were divided into: 1) chow diet, 2) HF, and 3) HF + NanoSOD. RESULTS: The HF + NanoSOD-treated mice showed a significant decrease in plasma and liver triglycerides when compared with HF-fed mice. Interestingly, NanoSOD reduced the expression of macrophage and inflammatory markers in visceral AT (VAT) and stromal cells derived from VAT. Moreover, the activation of proinflammatory signaling pathways, in particular, the extracellular signal-regulated kinases, was blunted in VAT on NanoSOD treatment. However, markers of oxidative stress were not altered significantly in the HF + NanoSOD group in the experimental conditions. Pretreatment of either macrophages or adipocytes significantly reduced the inflammatory response invoked in an in vitro coculture system, further supporting the role of NanoSOD in inhibiting obesity-linked inflammation. CONCLUSIONS: This data suggest that NanoSOD is effective not only in reducing AT macrophage accumulation and AT inflammation but also in promoting triglyceride metabolism in obesity.

Nanoformulated copper/zinc superoxide dismutase attenuates vascular cell activation and aortic inflammation in obesity.

OBJECTIVE: Endothelial cell (EC) oxidative stress can lead to vascular dysfunction which is an underlying event in the development of cardiovascular disease (CVD). The lack of a potent and bioavailable anti-oxidant enzyme is a major challenge in studies on antioxidant therapy. The objective of this study is to determine whether copper/zinc superoxide dismutase (CuZnSOD or SOD1) after nanoformulation (nanoSOD) can effectively reduce EC oxidative stress and/or vascular inflammation in obesity. METHODS: Human aortic endothelial cells (HAECs) were treated with native- or nanoSOD for 6 h followed by treatment with linoleic acid (LA), a free fatty acid, for 6-24 h. To determine the in vivo relevance, the effectiveness of nanoSOD in reducing vascular cell activation was studied in a mouse model of diet-induced obesity. RESULTS: We noted that nanoSOD was more effectively taken up by ECs than native SOD. Western blot analysis further confirmed that the intracellular accumulation of SOD1 protein was greatly increased upon nanoSOD treatment. Importantly, nanoSOD pretreatment led to a significant decrease in LA-induced oxidative stress in ECs which was associated with a marked increase in SOD enzyme activity in ECs. In vivo studies showed a significant decrease in markers of EC/vascular cell activation and/or inflammation in visceral adipose tissue (VAT), thoracic aorta, and heart collected from nanoSOD-treated mice compared to obese control mice. Interestingly, the expression of metallothionein 2, an antioxidant gene was significantly increased in nanoSOD-treated mice. CONCLUSION: Our data show that nanoSOD is very effective in delivering active SOD to ECs and in reducing EC oxidative stress. Our data also demonstrate that nanoSOD will be a useful tool to reduce vascular cell activation in VAT and aorta in obesity which, in turn, can protect against obesity-associated CVD, in particular, hypertension.

Hematopoietic cyclooxygenase-2 deficiency increases adipose tissue inflammation and adiposity in obesity.

OBJECTIVE: Adipose tissue (AT) macrophages mediate AT inflammation in obesity, and cyclooxygenase-2 (COX-2) is a major inflammatory gene. It was hypothesized that deletion of hematopoietic COX-2 will inhibit AT inflammation in obesity. METHODS: Lethally irradiated wild-type (WT) mice were injected with bone marrow (BM) cells collected from WT or COX-2 knock-out (COX-2-/-) donor mice and fed a high-fat diet for 16 weeks. RESULTS: The mice that received BM cells from COX-2-/- mice (BM-COX-2-/-) gained increased body weight, fat mass, and visceral AT (VAT) mass. These mice exhibited reduced inflammatory markers in the VAT stromal vascular cells (SVC). However, the inflammatory markers were increased in adipocyte fraction and/or whole VAT. The activation of ERK1/2 MAPK, a pro-inflammatory signaling pathway, was increased in BM-COX-2-/- mice. The molecular markers of adipogenesis were increased in the VAT or adipocyte fraction. Wnt signaling markers which inhibit adipogenesis, including Wnt3A and DVL3, were reduced, and Wnt5a/b which promotes inflammation was increased in the VAT and/or adipocytes. Finally, an increase in hepatic triglyceride levels in BM-COX-2-/- mice was noted. CONCLUSIONS: The data suggest that COX-2 deletion in hematopoietic cells reduces SVC inflammation but increases VAT inflammation and promotes adiposity likely via altered Wnt signaling.

Effects of leptin replacement alone and with exendin-4 on food intake and weight regain in weight-reduced diet-induced obese rats.

Weight loss in obese humans produces a relative leptin deficiency, which is postulated to activate potent orexigenic and energy conservation mechanisms to restrict weight loss and promote weight regain. Here we determined whether leptin replacement alone or with GLP-1 receptor agonist exendin-4 attenuates weight regain or promotes greater weight loss in weight-reduced diet-induced obese (DIO) rats. Forty percent restriction in daily intake of a high-fat diet in DIO rats for 4 wk reduced body weight by 12%, body fat by 29%, and plasma leptin by 67% and normalized leptin sensitivity. When food restriction ended, body weight, body fat, and plasma leptin increased rapidly. Daily administration of leptin [3-h intraperitoneal (ip) infusions (4 nmol·kg(-1)·h(-1))] at onset and end of dark period for 3 wk did not attenuate hyperphagia and weight regain, nor did it affect mean daily meal sizes or meal numbers. Exendin-4 (50 pmol·kg(-1)·h(-1)) infusions during the same intervals prevented postrestriction hyperphagia and weight regain by normalizing meal size. Coadministration of leptin and exendin-4 did not reduce body weight more than exendin-4 alone. Instead, leptin began to attenuate the inhibitory effects of exendin-4 on food intake, meal size, and weight regain by the end of the second week of administration. Plasma leptin in rats receiving leptin was sevenfold greater than in rats receiving vehicle and 17-fold greater than in rats receiving exendin-4. Together, these results do not support the hypothesis that leptin replacement alone or with exendin-4 attenuates weight regain or promotes greater weight loss in weight-reduced DIO rats.