Acetylsalicylic acid and salicylic acid present anticancer properties against melanoma by promoting nitric oxide-dependent endoplasmic reticulum stress and apoptosis.

Melanoma is the most aggressive and fatal type of skin cancer due to being highly proliferative. Acetylsalicylic acid (ASA; Aspirin) and salicylic acid (SA) are ancient drugs with multiple applications in medicine. Here, we showed that ASA and SA present anticancer effects against a murine model of implanted melanoma. These effects were also validated in 3D- and 2D-cultured melanoma B16F10 cells, where the drugs promoted pro-apoptotic effects. In both in vivo and in vitro models, SA and ASA triggered endoplasmic reticulum (ER) stress, which culminates with the upregulation of the pro-apoptotic transcription factor C/EBP homologous protein (CHOP). These effects are initiated by ASA/SA-triggered Akt/mTOR/AMPK-dependent activation of nitric oxide synthase 3 (eNOS), which increases nitric oxide and reactive oxygen species production inducing ER stress response. In the end, we propose that ASA and SA instigate anticancer effects by a novel mechanism, the activation of ER stress.

Insulin specifically regulates expression of liver and muscle phosphofructokinase isoforms.

Phosphofructokinase (PFK) is a key regulatory enzyme of glycolysis, being considered the pacemaker of this pathway. In mammals, this enzyme exists as three different isoforms, PFKM, PFKL and PFKP, presenting different regulatory and catalytic properties. The expression of these isoforms is tissue-specific and vary according to the cell differentiation and signalization. Although it is known that the expression of the different PFK isoforms directly affects cell function, the information regarding the regulation of PFK isoforms expression is scarce. In the present work, we evaluate the role of insulin signalization on the expression of three PFK isoforms on skeletal muscle, liver, and epididymal white adipose tissue (eWAT) of mice. For this, Swiss mice were treated with streptozotocin (STZ) to disrupt pancreatic ß-cells and, thus, insulin production. Control group were treated with citrate buffer (STZ vehicle). These groups were then treated with insulin or saline twice a day for ten consecutive days when animals were euthanized and tissues used for the evaluation of PFK isoforms expression by quantitative PCR (qPCR). Our results revealed that the lack of insulin significantly impacted the expression of PFKL, presenting mild effects on PFKM and no effects on PFKP. The decrease of PFKL and PFKM mRNA levels observed on the group treated with STZ was reversed by the treatment with insulin. In conclusion, insulin, the most known regulator of glucose consumption, specifically regulates the expression of PFKL and PFKM, which impact the regulation of glycolysis in the cell.

Effects of Food Additives on Immune Cells As Contributors to Body Weight Gain and Immune-Mediated Metabolic Dysregulation.

Food additives are compounds used in order to improve food palatability, texture, and shelf life. Despite a significant effort to assure safety of use, toxicological analysis of these substances, generally, rely on their direct toxicity to target organs (liver and kidney) or their genotoxic effects. Much less attention is paid to the effects of these compounds on cells of the immune system. This is of relevance given that metabolic dysregulation and obesity have a strong immune-mediated component. Obese individuals present a state of chronic low-grade inflammation that contributes to the establishment of insulin resistance and other metabolic abnormalities known as the metabolic syndrome. Obesity and metabolic syndrome are currently recognized as worldwide epidemics that pose a profound socioeconomic impact and represent a concern to public health. Cells of the immune system contribute to both the maintenance of "lean homeostasis" and the metabolic dysregulation observed in obese individuals. Although much attention has been drawn in the past decades to obesity and metabolic syndrome as a result of ingesting highly processed food containing large amounts of fat and simple sugars, mounting evidence suggest that food additives may also be important contributors to metabolic derangement. Herein, we review pieces of evidence from the literature showing that food additives have relevant effects on cells of the immune system that could contribute to immune-mediated metabolic dysregulation. Considering their potential to predispose individuals to develop obesity and metabolic syndrome, their use should be taken with caution or maybe revisited.

Reference genes for quantitative PCR in the adipose tissue of mice with metabolic disease.

Obesity and diabetes are metabolic diseases and they are increasing in prevalence. The dynamics of gene expression associated with these diseases is fundamental to identifying genes involved in related biological processes. qPCR is a sensitive technique for mRNA quantification and the most commonly used method in gene-expression studies. However, the reliability of these results is directly influenced by data normalization. As reference genes are the major normalization method used, this work aims to identify reference genes for qPCR in adipose tissues of mice with type-I diabetes or obesity. We selected 12 genes that are commonly used as reference genes. The expression of these genes in the adipose tissues of mice was analyzed in the context of three different experimental protocols: 1) untreated animals; 2) high-fat-diet animals; and 3) streptozotocin-treated animals. Gene-expression stability was analyzed using four different algorithms. Our data indicate that TATA-binding protein is stably expressed across adipose tissues in control animals. This gene was also a useful reference when the brown adipose tissues of control and obese mice were analyzed. The mitochondrial ATP synthase F1 complex gene exhibits stable expression in subcutaneous and perigonadal adipose tissue from control and obese mice. Moreover, this gene is the best reference for qPCR normalization in adipose tissue from streptozotocin-treated animals. These results show that there is no perfect stable gene suited for use under all experimental conditions. In conclusion, the selection of appropriate genes is a prerequisite to ensure qPCR reliability and must be performed separately for different experimental protocols.

Metformin reverses hexokinase and phosphofructokinase downregulation and intracellular distribution in the heart of diabetic mice.

Diabetes mellitus is characterized by hyperglycemia and its associated complications, including cardiomyopathy. Metformin, in addition to lowering blood glucose levels, provides cardioprotection for diabetic subjects. Glycolysis is essential to cardiac metabolism and its reduction may contribute to diabetic cardiomyopathy. Hexokinase (HK) and phosphofructokinase (PFK), rate-limiting enzymes of glycolysis, are downregulated in cardiac muscle from diabetic subjects, playing a central role on the decreased glucose utilization in the heart of diabetic subjects. Thus, the aim of this study was to determine whether metformin modulates heart HK and PFK from diabetic mice. Diabetes was induced by streptozotocin injection on male Swiss mice, which were treated for three consecutive days with 250 mg/kg metformin before evaluating HK and PFK activity, expression, and intracellular distribution on the heart of these subjects. We show that metformin abrogates the downregulation of HK and PFK in the heart of streptozotocin-induced diabetic mice. This effect is not correlated to alteration on the enzymes' transcription and expression. However, the intracellular distribution of both enzymes is altered in diabetic hearts that show increased activity of the soluble fraction when compared to the particulate fraction. Moreover, this pattern is reversed upon the treatment with metformin, which is correlated with the effects of the drug on the enzymes activity. Altogether, our results support evidences that metformin alter the intracellular localization of HK and PFK augmenting glucose utilization by diabetic hearts and, thus, conferring cardiac protection to diabetic subjects.