Intervention with citrus flavonoids reverses obesity and improves metabolic syndrome and atherosclerosis in obese Ldlr \u3csup\u3e/\u3c/sup\u3e mice

Copyright © 2018 Burke et al. Obesity and its associated metabolic dysfunction and cardiovascular disease risk represent a leading cause of adult morbidity worldwide. Currently available pharmacological therapies for obesity have had limited success in reversing existing obesity and metabolic dysregulation. Previous prevention studies demonstrated that the citrus flavonoids, naringenin and nobiletin, protect against obesity and metabolic dysfunction in Ldlr/ mice fed a high-fat cholesterol-containing (HFHC) diet. However, their effects in an intervention model are unknown. In this report, we show that, in Ldlr/ mice with diet-induced obesity, citrus flavonoid supplementation to a HFHC diet reversed existing obesity and adipocyte size and number through enhanced energy expenditure and increased hepatic fatty acid oxidation. Caloric intake was unaffected and no evidence of white adipose tissue browning was observed. Reversal of adiposity was accompanied by improvements in hyperlipidemia, insulin sensitivity, hepatic steatosis, and a modest reduction in blood monocytes. Together, this resulted in atherosclerotic lesions that were unchanged in size, but characterized by reduced macrophage content, consistent with a more stable plaque phenotype. These studies further suggest potential therapeutic utility of citrus flavonoids, especially in the context of existing obesity, metabolic dysfunction, and cardiovascular disease

Peroxisome proliferator-activated receptor δ agonist GW1516 attenuates diet-induced aortic inflammation, insulin resistance, and atherosclerosis in low-density lipoprotein receptor knockout mice

OBJECTIVE - The peroxisome proliferator-activated receptor (PPAR) δ regulates systemic lipid homeostasis and inflammation. However, the ability of PPARδ agonists to improve the pathology of pre-established lesions and whether PPARδ activation is atheroprotective in the setting of insulin resistance have not been reported. Here, we examine whether intervention with a selective PPARδ agonist corrects metabolic dysregulation and attenuates aortic inflammation and atherosclerosis. APPROACH AND RESULTS - Low-density lipoprotein receptor knockout mice were fed a chow or a high-fat, high-cholesterol (HFHC) diet (42% fat, 0.2% cholesterol) for 4 weeks. For a further 8 weeks, the HFHC group was fed either HFHC or HFHC plus GW1516 (3 mg/kg per day). GW1516 significantly attenuated pre-established fasting hyperlipidemia, hyperglycemia, and hyperinsulinemia, as well as glucose and insulin intolerance. GW1516 intervention markedly reduced aortic sinus lesions and lesion macrophages, whereas smooth muscle α-actin was unchanged and collagen deposition enhanced. In aortae, GW1516 increased the expression of the PPARδ-specific gene Adfp but not PPARα- or γ-specific genes. GW1516 intervention decreased the expression of aortic proinflammatory M1 cytokines, increased the expression of the anti-inflammatory M2 cytokine Arg1, and attenuated the iNos/Arg1 ratio. Enhanced mitogen-activated protein kinase signaling, known to induce inflammatory cytokine expression in vitro, was enhanced in aortae of HFHC-fed mice. Furthermore, the HFHC diet impaired aortic insulin signaling through Akt and forkhead box O1, which was associated with elevated endoplasmic reticulum stress markers CCAAT-enhancer-binding protein homologous protein and 78kDa glucose regulated protein. GW1516 intervention normalized mitogen-activated protein kinase activation, insulin signaling, and endoplasmic reticulum stress. CONCLUSIONS - Intervention with a PPARδ agonist inhibits aortic inflammation and attenuates the progression of pre-established atherosclerosis. © 2013 American Heart Association, Inc

Two-week isocaloric time-restricted feeding decreases liver inflammation without significant weight loss in obese mice with non-alcoholic fatty liver disease

Prolonged, isocaloric, time-restricted feeding (TRF) protocols can promote weight loss, improve metabolic dysregulation, and mitigate non-alcoholic fatty liver disease (NAFLD). In addition, 3-day, severe caloric restriction can improve liver metabolism and glucose homeostasis prior to significant weight loss. Thus, we hypothesized that short-term, isocaloric TRF would improve NAFLD and characteristics of metabolic syndrome in diet-induced obese male mice. After 26 weeks of ad libitum access to western diet, mice either continued feeding ad libitum or were provided with access to the same quantity of western diet for 8 h daily, over the course of two weeks. Remarkably, this short-term TRF protocol modestly decreased liver tissue inflammation in the absence of changes in body weight or epidydimal fat mass. There were no changes in hepatic lipid accumulation or other characteristics of NAFLD. We observed no changes in liver lipid metabolism-related gene expression, despite increased plasma free fatty acids and decreased plasma triglycerides in the TRF group. However, liver Grp78 and Txnip expression were decreased with TRF suggesting hepatic endoplasmic reticulum (ER) stress and activation of inflammatory pathways may have been diminished. We conclude that two-week, isocaloric TRF can potentially decrease liver inflammation, without significant weight loss or reductions in hepatic steatosis, in obese mice with NAFLD

Microarray data and pathway analyses for primary human activated hepatic stellate cells compared to HepG2 human hepatoma cells

As nonalcoholic fatty liver disease progresses to end-stage diseases, including fibrosis, cirrhosis and hepatocellular carcinoma, fibrotic activated hepatic stellate cells and cancerous epithelial cells can become abundant, changing the cellular composition of this organ. Despite potentially residing within the same diseased tissue, direct comparisons of global gene expression between activated hepatic stellate cells and hepatocellular carcinoma cells are lacking. Here we provide data collected using Affymetrix GeneChip microarrays to identify differential gene expression in cultured primary human activated hepatic stellate cells compared to HepG2 human hepatoma cells. The dataset includes many genes involved in intermediary metabolism which were investigated in greater depth in our associated article (A.M. Hetherington, C.G. Sawyez, E. Zilberman, A.M. Stoianov, D.L. Robson, J.M. Hughes-Large, et al., 2016) [1]. Pathway analyses of known protein coding genes down-regulated or up-regulated by greater than 2.0-fold are also provided

Elongation Factor 1A-1 Is a Mediator of Hepatocyte Lipotoxicity Partly through Its Canonical Function in Protein Synthesis.

Elongation factor 1A-1 (eEF1A-1) has non-canonical functions in regulation of the actin cytoskeleton and apoptosis. It was previously identified through a promoter-trap screen as a mediator of fatty acid-induced cell death (lipotoxicity), and was found to participate in this process downstream of ER stress. Since ER stress is implicated in the pathogenesis of nonalcoholic fatty liver disease (NAFLD), we investigated the mechanism of action of eEF1A-1 in hepatocyte lipotoxicity. HepG2 cells were exposed to excess fatty acids, followed by assessments of ER stress, subcellular localization of eEF1A-1, and cell death. A specific inhibitor of eEF1A-1 elongation activity, didemnin B, was used to determine whether its function in protein synthesis is involved in lipotoxicity. Within 6 h, eEF1A-1 protein was modestly induced by high palmitate, and partially re-localized from its predominant location at the ER to polymerized actin at the cell periphery. This early induction and subcellular redistribution of eEF1A-1 coincided with the onset of ER stress, and was later followed by cell death. Didemnin B did not prevent the initiation of ER stress by high palmitate, as indicated by eIF2α phosphorylation. However, consistent with sustained inhibition of eEF1A-1-dependent elongation activity, didemnin B prevented the recovery of protein synthesis and increase in GRP78 protein that are normally associated with later phases of the response to ongoing ER stress. This resulted in decreased palmitate-induced cell death. Our data implicate eEF1A-1, and its function in protein synthesis, in hepatocyte lipotoxicity

Differential Lipotoxic Effects of Palmitate and Oleate in Activated Human Hepatic Stellate Cells and Epithelial Hepatoma Cells

Background/Aims: Nonalcoholic fatty liver disease (NAFLD) progression to fibrosis, cirrhosis and hepatocellular carcinoma, alters the cellular composition of this organ. During late-stage NAFLD, fibrotic and possibly cancerous cells can proliferate and, like normal hepatocytes, are exposed to high concentrations of fatty acids from both surrounding tissue and circulating lipid sources. We hypothesized that primary human activated hepatic stellate cells and epithelial hepatoma (HepG2) cells respond differently to lipotoxic conditions, and investigated the mechanisms involved. Methods: Primary activated hepatic stellate cells and HepG2 cells were exposed to pathophysiological concentrations of fatty acids and comparative studies of lipid metabolic and stress response pathways were performed. Results: Both cell types remained proliferative during exposure to a combination of palmitate plus oleate reflective of the general saturated versus unsaturated fatty acid composition of western diets. However, exposure to either high palmitate or high oleate alone induced cytotoxicity in activated stellate cells, while only palmitate caused cytotoxicity in HepG2 cells. mRNA microarray and biochemical comparisons revealed that stellate cells stored markedly less fatty acids as neutral lipids, and had reduced capacity for beta-oxidation. Similar to previous observations in HepG2 cells, palmitate, but not oleate, induced ER stress and actin stress fiber formation in activated stellate cells. In contrast, oleate, but not palmitate, induced the inflammatory signal TXNIP, decreased cytoskeleton proteins, and decreased cell polarity preceding cell death in activated stellate cells. Conclusions: Palmitate-induced lipotoxicity was associated with ER stress pathways in both primary activated hepatic stellate cells and epithelial hepatoma cells, whereas high oleate caused lipotoxicity only in activated stellate cells, possibly through a distinct mechanism involving disruption of cytoskeleton components. This may have implications for optimal dietary fatty acid compositions during various stages of NAFLD

Chemical Inhibition of eEF1A-1 Peptide Elongation Activity Decreases Palmitate-Induced Cell Death.

<p>(A) HepG2 cells were treated for 48 h with didemnin B (DB), followed by assessment of total protein synthesis by [³H] leucine incorporation. The IC₅₀ for protein synthesis was 80 nM. (B) Cells were incubated as in A, followed by assessment of cellular metabolism by MTT reduction. (C, D) HepG2 cells were incubated for 6 h with growth media containing BSA or 1.0 mM palmitate (PA), as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131269#pone.0131269.g001" target="_blank">Fig 1</a>, in the presence or absence of 80 nM didemnin (DB). Phosphorylated and total eIF2α, and GRP78 proteins were detected in whole cell lysates by immunoblotting. Bands were quantified by densitometry and normalized to total eIF2α or actin, as indicated. Representative blots are shown. Black vertical lines on the blots in D indicate that lanes from the same blot were re-ordered for presentation purposes. (E) Cells were incubated for the times indicated with growth media containing BSA or 1.0 mM PA, in the presence or absence of 80 nM DB, followed by assessment of total protein synthesis by [³H] leucine incorporation. (F) HepG2 cells were incubated for 48 h with PA, in the presence or absence of 80 nM didemnin (DB). Cells were harvested and stained with PI and AnnV. Percentages of apoptotic and dead cells were determined by flow cytometry. For A and B, data are percentages of control (vehicle) ± SEM for n = 3. For C, D, E, and F, data are means ± SEM for n = 4. Different lower case letters are statistically significant at p < 0.05.</p

Parameters of Metabolic Disease and NAFLD in a Mouse Model of Obesity.

<p>Six week old male C57BL/6J mice and leptin-deficient (<i>ob/ob</i>) mice were maintained on semi-purified diet for 4 weeks. Data are means ± SEM, for n = 8.</p><p>* p < 0.05 for <i>ob/ob</i> mice compared to wild-type C57BL/6J mice.</p><p>Parameters of Metabolic Disease and NAFLD in a Mouse Model of Obesity.</p

eEF1A-1 Protein is Modestly Increased in HepG2 Cells During Fatty Acid-Induced ER stress, Preceding Cell Death.

<p>(A) HepG2 cells were incubated for 6 h with growth media containing increasing concentrations of BSA alone, palmitate (PA), oleate (OA) or a combination of palmitate and oleate (2:3 ratio, PA:OA). All fatty acids were conjugated to fatty acid free BSA at a molar ratio of 2:1. eEF1A-1, GRP78, and actin proteins were detected in whole cell lysates by immunoblotting. Representative blots are shown. (B, C) Immunoblots from experiments performed as described for A were quantified by densitometry. Signals for eEF1A-1 and GRP78 were normalized to actin. (D) HepG2 cells were incubated for 48 h with growth media containing BSA alone, or 1.0 mM fatty acids as in A. Cells were harvested, stained with propidium iodide, and the proportions of dead cells were determined by flow cytometry. All data are means ± SEM for n = 4–8. For B and C different lower case letters are statistically significant at p<0.05 for different concentrations within treatments (BSA or fatty acids).</p