Helena C. F. Oliveira

Saturated Fatty Acids Produce an Inflammatory Response Predominantly through the Activation of TLR4 Signaling in Hypothalamus: Implications for the Pathogenesis of Obesity

In animal models of diet-induced obesity, the activation of an inflammatory response in the hypothalamus produces molecular and functional resistance to the anorexigenic hormones insulin and leptin. The primary events triggered by dietary fats that ultimately lead to hypothalamic cytokine expression and inflammatory signaling are unknown. Here, we test the hypothesis that dietary fats act through the activation of toll-like receptors 2/4 and endoplasmic reticulum stress to induce cytokine expression in the hypothalamus of rodents. According to our results, long-chain saturated fatty acids activate predominantly toll-like receptor 4 signaling, which determines not only the induction of local cytokine expression but also promotes endoplasmic reticulum stress. Rats fed on a monounsaturated fat-rich diet do not develop hypothalamic leptin resistance, whereas toll-like receptor 4 loss-of-function mutation and immunopharmacological inhibition of toll-like receptor 4 protects mice from diet-induced obesity. Thus, toll-like receptor 4 acts as a predominant molecular target for saturated fatty acids in the hypothalamus, triggering the intracellular signaling network that induces an inflammatory response, and determines the resistance to anorexigenic signals.

Fatty acid synthase inhibition with Orlistat promotes apoptosis and reduces cell growth and lymph node metastasis in a mouse melanoma model

Fatty acid synthase (FASN) is the enzyme responsible for the endogenous synthesis of the saturated fatty acid palmitate. In contrast to most normal cells, malignant cells depend on FASN activity for growth and survival. In fact, FASN is overexpressed in a variety of human cancers including cutaneous melanoma, in which its levels of expression are associated with a poor prognosis and depth of invasion. Here, we show that the specific inhibition of FASN activity by the antiobesity drug Orlistat or siRNA is able to significantly reduce proliferation and promote apoptosis in the mouse metastatic melanoma cell line B16‐F10. These results prompted us to verify the effect of FASN inhibition on the metastatic process in a model of spontaneous melanoma metastasis, in which B16‐F10 cells injected in the peritoneal cavity of C57BL/6 mice metastasize to the mediastinal lymph nodes. We observed that mice treated with Orlistat 48 hr after the inoculation of B16‐F10 cells exhibited a 52% reduction in the number of mediastinal lymph node metastases, in comparison with the control animals. These results suggest that FASN activity is essential for B16‐F10 melanoma cell proliferation and survival while its inactivation by Orlistat significantly reduces their metastatic spread. The chemical inhibition of FASN activity could have a potential benefit in association with the current chemotherapy for melanoma.

Oxidative stress in atherosclerosis-prone mouse is due to low antioxidant capacity of mitochondria

Atherosclerotic disease remains a leading cause of death in westernized societies, and reactive oxygen species (ROS) play a pivotal role in atherogenesis. Mitochondria are the main intracellular sites of ROS generation and are also targets for oxidative damage. Here, we show that mitochondria from atherosclerosis‐prone, hypercholesterolemic low‐density lipoprotein (LDL) receptor knockout mice have oxidative phosphorylation efficiency similar to that from control mice but have a higher net production of ROS and susceptibility to develop membrane permeability transition. Increased ROS production was observed in mitochondria isolated from several tissues, including liver, heart, and brain, and in intact mononuclear cells from spleen. In contrast to control mitochondria, knockout mouse mitochondria did not sustain a reduced state of matrix NADPH, the main source of antioxidant defense against ROS. Experiments in vivo showed faster liver secretion rates and de novo synthesis of triglycerides and cholesterol in knockout than in control mice, suggesting that increased lipogenesis depleted the reducing equivalents from NADPH and generated a state of oxidative stress in hypercholesterolemic knockout mice. These data provide the first evidence of how oxidative stress is generated in LDL receptor defective cells and could explain the increased LDL oxidation, cell death, and atherogenesis seen in familiar hypercholesterolemia.

A spontaneous mutation in the nicotinamide nucleotide transhydrogenase gene of C57BL/6J mice results in mitochondrial redox abnormalities.

NADPH is the reducing agent for mitochondrial H2O2 detoxification systems. Nicotinamide nucleotide transhydrogenase (NNT), an integral protein located in the inner mitochondrial membrane, contributes to an elevated mitochondrial NADPH/NADP(+) ratio. This enzyme catalyzes the reduction of NADP(+) at the expense of NADH oxidation and H(+) reentry to the mitochondrial matrix. A spontaneous Nnt mutation in C57BL/6J (B6J-Nnt(MUT)) mice arose nearly 3 decades ago but was only discovered in 2005. Here, we characterize the consequences of the Nnt mutation on the mitochondrial redox functions of B6J-Nnt(MUT) mice. Liver mitochondria were isolated both from an Nnt wild-type C57BL/6 substrain (B6JUnib-Nnt(W)) and from B6J-Nnt(MUT) mice. The functional evaluation of respiring mitochondria revealed major redox alterations in B6J-Nnt(MUT) mice, including an absence of transhydrogenation between NAD and NADP, higher rates of H2O2 release, the spontaneous oxidation of NADPH, the poor ability to metabolize organic peroxide, and a higher susceptibility to undergo Ca(2+)-induced mitochondrial permeability transition. In addition, the mitochondria of B6J-Nnt(MUT) mice exhibited increased oxidized/reduced glutathione ratios as compared to B6JUnib-Nnt(W) mice. Nonetheless, the maximal activity of NADP-dependent isocitrate dehydrogenase, which is a coexisting source of mitochondrial NADPH, was similar between both groups. Altogether, our data suggest that NNT functions as a high-capacity source of mitochondrial NADPH and that its functional loss due to the Nnt mutation results in mitochondrial redox abnormalities, most notably a poor ability to sustain NADP and glutathione in their reduced states. In light of these alterations, the potential drawbacks of using B6J-Nnt(MUT) mice in biomedical research should not be overlooked.

The Fatty Acid Synthase Inhibitor Orlistat Reduces the Growth and Metastasis of Orthotopic Tongue Oral Squamous Cell Carcinomas

Fatty acid synthase (FASN) is the biosynthetic enzyme responsible for the endogenous synthesis of fatty acids. It is downregulated in most normal cells, except in lipogenic tissues such as liver, lactating breast, fetal lung, and adipose tissue. Conversely, several human cancers, including head and neck squamous cell carcinomas (HNSCC), overexpress FASN, which has been associated with poor prognosis and recently suggested as a metabolic oncoprotein. Orlistat is an irreversible inhibitor of FASN activity with cytotoxic properties on several cancer cell lines that inhibits tumor progression and metastasis in prostate cancer xenografts and experimental melanomas, respectively. To explore whether the inhibition of FASN could impact oral tongue squamous cell carcinoma (OTSCC) metastatic spread, an orthotopic model was developed by the implantation of SCC-9 ZsGreen LN-1 cells into the tongue of BALB/c nude mice. These cells were isolated through in vivo selection, show a more invasive behavior in vitro than the parental cells, and generate orthotopic tumors that spontaneously metastasize to cervical lymph nodes in 10 to 15 days only. SCC-9 ZsGreen LN-1 cells also exhibit enhanced production of MMP-2, ERBB2, and CDH2. The treatment with orlistat reduced proliferation and migration, promoted apoptosis, and stimulated the secretion of VEGFA165b by SCC-9 ZsGreen LN-1 cells. In vivo, the drug was able to decrease both the volume and proliferation indexes of the tongue orthotopic tumors and, importantly, reduced the number of metastatic cervical lymph nodes by 43%. These results suggest that FASN is a potential molecular target for the chemotherapy of patients with OTSCC. Mol Cancer Ther; 13(3); 585–95. ©2013 AACR.

Inhibition of fatty acid synthase in melanoma cells activates the intrinsic pathway of apoptosis

Fatty acid synthase (FASN) is the metabolic enzyme responsible for the endogenous synthesis of the saturated long-chain fatty acid, palmitate. In contrast to most normal cells, FASN is overexpressed in a variety of human cancers, including cutaneous melanoma, in which its levels of expression are associated with tumor invasion and poor prognosis. We have previously shown that FASN inhibition with orlistat significantly reduces the number of spontaneous mediastinal lymph node metastases following the implantation of B16-F10 mouse melanoma cells in the peritoneal cavity of C57BL/6 mice. In this study, we investigate the biological mechanisms responsible for the FASN inhibition-induced apoptosis in B16-F10 cells. Both FASN inhibitors, cerulenin and orlistat, significantly reduced melanoma cell proliferation and activated the intrinsic pathway of apoptosis, as demonstrated by the cytochrome c release and caspase-9 and -3 activation. Further, apoptosis was preceded by an increase in both reactive oxygen species production and cytosolic calcium concentrations and independent of p53 activation and mitochondrial permeability transition. Taken together, these findings demonstrate the mitochondrial involvement in FASN inhibition-induced apoptosis in melanoma cells.

Sex differences in risk factors for coronary heart disease: a study in a Brazilian population

BackgroundIn Brazil coronary heart disease (CHD) constitutes the most important cause of death in both sexes in all the regions of the country and interestingly, the difference between the sexes in the CHD mortality rates is one of the smallest in the world because of high rates among women. Since a question has been raised about whether or how the incidence of several CHD risk factors differs between the sexes in Brazil the prevalence of various risk factors for CHD such as high blood cholesterol, diabetes mellitus, hypertension, obesity, sedentary lifestyle and cigarette smoking was compared between the sexes in a Brazilian population; also the relationships between blood cholesterol and the other risk factors were evaluated.ResultsThe population presented high frequencies of all the risk factors evaluated. High blood cholesterol (CHOL) and hypertension were more prevalent among women as compared to men. Hypertension, diabetes and smoking showed equal or higher prevalence in women in pre-menopausal ages as compared to men. Obesity and physical inactivity were equally prevalent in both sexes respectively in the postmenopausal age group and at all ages. CHOL was associated with BMI, sex, age, hypertension and physical inactivity.ConclusionsIn this population the high prevalence of the CHD risk factors indicated that there is an urgent need for its control; the higher or equal prevalences of several risk factors in women could in part explain the high rates of mortality from CHD in females as compared to males.

Mangifera indica L. extract (Vimang ® ) and its main polyphenol mangiferin prevent mitochondrial oxidative stress in atherosclerosis-prone hypercholesterolemic mouse

Atherosclerosis is linked to a number of oxidative events ranging from low-density lipoprotein (LDL) oxidation to the increased production of intracellular reactive oxygen species (ROS). We have recently demonstrated that liver mitochondria isolated from the atherosclerosis-prone hypercholesterolemic LDL receptor knockout (LDLr(-/-)) mice have lower content of NADP(H)-linked substrates than the controls and, as consequence, higher sensitivity to oxidative stress and mitochondrial membrane permeability transition (MPT). In the present work, we show that oral supplementation with the antioxidants Mangifera indica L. extract (Vimang) or its main polyphenol mangiferin shifted the sensitivity of LDLr(-/-) liver mitochondria to MPT to control levels. These in vivo treatments with Vimang and mangiferin also significantly reduced ROS generation by both isolated LDLr(-/-) liver mitochondria and spleen lymphocytes. In addition, these antioxidant treatments prevented mitochondrial NAD(P)H-linked substrates depletion and NADPH spontaneous oxidation. In summary, Vimang and mangiferin spared the endogenous reducing equivalents (NADPH) in LDLr(-/-) mice mitochondria correcting their lower antioxidant capacity and restoring the organelle redox homeostasis. The effective bioavailability of these compounds makes them suitable antioxidants with potential use in atherosclerosis susceptible conditions.

Primary hypercholesterolaemia impairs glucose homeostasis and insulin secretion in low-density lipoprotein receptor knockout mice independently of high-fat diet and obesity.

We investigated whether primary hypercholesterolaemia per se affects glucose homeostasis and insulin secretion in low-density lipoprotein receptor knockout mice (LDLR(-/-)). Glucose plasma levels were increased and insulin decreased in LDLR(-/-) compared to the wild-type mice. LDLR(-/-) mice presented impaired glucose tolerance, but normal whole body insulin sensitivity. The dose-response curve of glucose-stimulated insulin secretion was shifted to the right in LDLR(-/-) islets. Significant reductions in insulin secretion in response to l-leucine or 2-ketoisocaproic acid were also observed in LDLR(-/-). Islet morphometric parameters, total insulin and DNA content were similar in both groups. Glucose uptake and oxidation were reduced in LDLR(-/-) islets. Removal of cholesterol from LDLR(-/-) islets corrected glucose-stimulated insulin secretion. These results indicate that enhanced membrane cholesterol content due to hypercholesterolaemia leads to a lower insulin secretion and glucose intolerance without affecting body insulin sensitivity. This represents an additional risk factor for diabetes and atherosclerosis in primary hypercholesterolaemia.

Cholesteryl ester transfer protein: The controversial relation to atherosclerosis and emerging new biological roles

Cholesteryl ester transfer protein (CETP) exerts a profound impact on high‐density lipoprotein (HDL) metabolism and, consequently, on the risk of atherosclerosis development and cardiovascular mortality. Here, we review the complex relationship between CETP and atherosclerosis based upon the experimental, clinical, and epidemiological studies. In addition, we discuss the recent findings that expand the functions of CETP to new areas of interest such as Alzheimers disease, inflammation, and obesity.