1. Investigación

The aim of this study was to evaluate the effect of Compound 21 (C21), a selective AT2R agonist, on the prevention of endothelial dysfunction, extracellular matrix (ECM) remodeling and arterial stiffness associated with diet-induced obesity (DIO). 5-week-old male C57BL/6J mice were fed a standard (Chow) or high-fat diet (HF) for 6 weeks.Half of the animals of each group were simultaneously treated with C21 (1mg/kg/day, in the drinking water), generating 4 groups: Chow C, Chow C21, HF C, HF C21. Vascular function and mechanical properties were determined in the abdominal aorta. To evaluate ECM remodeling, collagen deposition, activity of metalloproteinases (MMP) 2 and 9 and TGF-1 concentration were analyzed in the plasma. Abdominal aortas from HF C mice showed endothelial dysfunction as well as enhanced contractile but reduced relaxant responses to Ang II.This effect was abrogated with C21 treatment by preserving NO availability. A left-shift inthe tension-stretch relationship, paralleled by an augmented β-index (marker of intrinsic arterial stiffness), and enhanced collagen deposition and MMP-2/-9 activities were also detected in HF mice. However, when treated with C21, HF mice exhibited lower TGF-1 levels in abdominal aortas together with reduced MMP activities and collagen deposition compared with HF C mice. In conclusion, these data demonstrate that AT2R stimulation by C21 in obesity preserves NO availability and prevents unhealthy vascular remodeling, thus protecting the abdominal aorta in HF mice against the development of endothelial dysfunction, ECM remodeling and arterial stiffness.

Brown adipose tissue (BAT) dissipates energy to produce heat. Thus, it has the potential to regulate body temperature by thermogenesis. For the last decade, BAT has been in the spotlight due to its rediscovery in adult humans. This is evidenced by over a hundred clinical trials that are currently registered to target BAT as a therapeutic tool in the treatment of metabolic diseases, such as obesity or diabetes. The goal of most of these trials is to activate the BAT thermogenic program via several approaches such as adrenergic stimulation, natriuretic peptides, retinoids, capsinoids, thyroid hormones, or glucocorticoids. However, the impact of BAT activation on total body energy consumption and the potential effect on weight loss is still limited. Other studies have focused on increasing the mass of thermogenic BAT. This can be relevant in obesity, where the activity and abundance of BAT have been shown to be drastically reduced. The aim of this review is to describe pathological processes associated with obesity that may influence the correct differentiation of BAT, such as catecholamine resistance, inflammation, oxidative stress, and endoplasmic reticulum stress. This will shed light on the thermogenic potential of BAT as a therapeutic approach to target obesity-induced metabolic diseases.

Congenital malformations are a common adverse outcome in pregnancies complicated by pregestational obesity, although the underlying mechanisms are still unrevealed. Our aim was to study the effect of oxidative stress in obesity-induced teratogenesis. Wistar rats were fed a high-fat diet for 13 weeks, with (OE group) or without (O group) vitamin E supplementation. Then, rats were mated and sacrificed at day 11.5 of gestation. Embryos from O dams presented a 25.9 3.5% rate of malformations (vs. 8.7 3.4% in C rats), which was reduced in the OE group (11.5 2.3%). Pregestational obesity induced hepatic protein and DNA oxidation and a decline in antioxidant enzymes. Importantly, glutathione content was also decreased, limiting the availability of this antioxidant in the embryos. Vitamin E supplementation efficiently maintained glutathione levels in the obese mothers, which could be used in their embryos to prevent oxidation-induced malformations. To test the effect of decreasing glutathione levels alone in a cell culture model of neuroepithelium, murine embryonic stem cells (ESC) were induced to form neuronal precursors and glutathione synthesis was inhibited with the gamma–glutamylcysteine synthesis inhibitor, buthionine sulfoximine (BSO). BSO inhibited the expression of Pax3, a gene required for neural tube closure that is also inhibited by oxidative stress. Taken together, our data indicate that obesity causes malformations through the depletion of maternal glutathione, thereby decreasing glutathione-dependent free radical scavenging in embryos, which can be prevented by vitamin E supplementation.

The rediscovery of brown adipose tissue (BAT) in humans and its capacity to oxidize fat and dissipate energy as heat has put the spotlight on its potential as a therapeutic target in the treatment of several metabolic conditions including obesity and diabetes. To date the measurement of bioenergetics parameters has required the use of cultured cells or extracted mitochondria with the corresponding loss of information in the tissue context. Herein, we present a method to quantify mitochondrial bioenergetics directly in BAT. Based on XF Seahorse Technology, we assessed the appropriate weight of the explants, the exact concentration of each inhibitor in the reaction, and the specific incubation time to optimize bioenergetics measurements. Our results show that BAT basal oxygen consumption is mostly due to proton leak. In addition, BAT presents higher basal oxygen consumption than white adipose tissue and a positive response to b-adrenergic stimulation. Considering the whole tissue and not just subcellular populations is a direct approach that provides a realistic view of physiological respiration. In addition, it can be adapted to analyze the effect of potential activators of thermogenesis, or to assess the use of fatty acids or glucose as a source of energy.

Caloric restriction (CR) improves endothelial function through the upregulation of adenosine monophosphateactivated protein kinase (AMPK) and endothelial nitric oxide synthase (eNOS). Moreover, hydrogen peroxide (H2O2) is upregulated in yeast subjected to CR. Our aim was to assess if mild short-term CR increases vascular H2O2 formation as a link with AMPK and eNOS activation. Twelve-week old Zucker obese (fa/fa) and control Zucker lean male rats were fed a standard chow either ad libitum (AL, n=10) or with a 20% CR (CR, n=10) for two weeks. CR significantly improved relaxation to ACh in fa/fa rats because of an enhanced endogenous production of H2O2 in aortic rings (H2O2 levels fa/faAL=0.5±0.05 nmol/mg vs. H2O2 levels fa/faCR=0.76±0.07 nmol/mg protein; p<0.05). Expression of mitochondrial superoxide dismutase (Mn-SOD) and total SOD activity were increased in aorta from fa/fa animals after CR. In cultured aortic endothelial cells, serum deprivation or 2-deoxy-D-glucose induced a significant increase in: i) superoxide anion and H2O2 levels, ii) p-AMPK/AMPK and p-eNOS/eNOS expression and iii) nitric oxide levels. This effect was reduced by catalase and strongly inhibited by Ca2+/calmodulin-dependent kinase II (CamkII) silencing. In conclusion, we propose that mild short-term CR might be a trigger of mechanisms aimed at protecting the vascular wall by the increase of H2O2, which then activates AMPK and nitric oxide release, thus improving endothelium-dependent relaxation. In addition, we demonstrate that CAMKII plays a key role in mediating CRinduced AMPK activation through H2O2 increase.

Obesity is associated with severe metabolic diseases such as type 2 diabetes, insulin resistance, cardiovascular disease and some forms of cancer. The pathophysiology of obesity-induced metabolic diseases has been strongly related to white adipose tissue (WAT) dysfunction through several mechanisms such as fibrosis, apoptosis, inflammation, ER and oxidative stress. However, little is known of whether these processes are also present in brown adipose tissue (BAT) during obesity, and the potential consequences on mitochondrial activity. Here we characterized the BAT of obese and hyperglycemic mice treated with a high-fat diet (HFD) for 20 weeks. The hypertrophic BAT from obese mice showed no signs of fibrosis nor apoptosis, but higher levels of inflammation, ER stress, ROS generation and antioxidant enzyme activity than the lean counterparts. The response was attenuated compared with obesity-induced WAT derangements, which suggests that BAT is more resistant to the obesity-induced insult. In fact, mitochondrial respiration in BAT from obese mice was enhanced, with a 2-fold increase in basal oxygen consumption, through the upregulation of complex III of the electron transport chain and UCP1. Altogether, our results show that obesity is accompanied by an increase in BAT mitochondrial activity, inflammation and oxidative damage.

The activation of endoplasmic reticulum (ER) stress and a reduction of AMP-dependent protein kinase (AMPK) phosphorylation have been described in obesity. We hypothesize that a moderate caloric restriction (CR) might contribute to reducing ER stress and increasing AMPK phosphorylation in peripheral tissues from genetically obese Zucker fa/fa rats and in peripheral blood mononuclear cells (PBMCs). Zucker Lean and Zucker fa/fa rats were fed with chow diet either ad libitum (AL) (C, as controls) or 80% of AL (CR) for 2 weeks, giving rise to four experimental groups: Lean C, Lean CR, fa/fa C and fa/fa CR. CR significantly increased AMPK phosphorylation in the liver, perirenal adipose tissue (PRAT) and PBMCs from fa/fa rats but not in the subcutaneous AT (SCAT), suggesting a reduced response of SCAT to CR. Liver samples of fa/fa rats exhibited an increased mRNA expression of PERK, EIF-2α, XBP-1(s), Chop and caspase 3, which was significantly reduced by CR. PRAT exhibited an overexpression of Edem and PDIA-4 in fa/fa rats, but only PDIA-4 expression was reduced by CR. eIF-2α phosphorylationwas significantly increased in all studied tissues fromfa/fa rats and reduced by CR. A negative correlationwas detected between p-AMPK and p-eIF-2α in the liver, PRAT and PBMCs from fa/fa rats but not in SCAT. This study shows that a moderate CR reduces ER stress and improves AMPK phosphorylation in several peripheral tissues and in circulating PBMCs, suggesting that alterations observed in PBMCs could reflect metabolic alterations associated with obesity. © 2020 Elsevier Inc. All rights reserved.