Characterization of Potential Intoxications with Medicines in a Regional Setting.

The Portuguese Poison Information Center (from Portuguese-CIAV) is a call center that offers medical assistance in case of possible intoxication with any kind of product, including medicines. This center´s main goal is to inform and guide the general public and health professionals. This work aimed to analyze and compare data corresponding to the telephone calls from the Algarve region (South of Portugal), received by CIAV during 2019 and 2020, regarding potential intoxications with medicines. To this end, data provided by CIAV on possible cases of medication intoxication in the Algarve region were collected, including the number of calls received, the place of origin of the call, the age group and sex of the intoxicated individual, the route of exposure to the drug, the circumstances of contact with the substance, the existence of symptoms, and the drug or drugs involved in the potential intoxication. The results showed that the number of cases slightly decreased in 2020 (n = 1261) compared with 2019 (n = 1340), with a high number of cases of intoxication in children between one and four years old in both years (21.2%; n = 152 in 2019; 16.4%; n = 115 in 2020). The drugs belonging to the locomotor system group (paracetamol and ibuprofen) were the main drugs involved, followed by the central nervous system pharmacotherapeutic group, namely benzodiazepines (diazepam and alprazolam). Paracetamol was the main drug responsible for the calls to CIAV (n = 71 in 2019; n = 63 in 2020), while for the remaining drugs there were fluctuations in their positions between both years. In some cases, this swinging may be explained by the possible changes in therapy due to potential interactions with drugs used for the treatment of symptoms of COVID-19 or perhaps related to misleading information released by the media about the use of some drugs, such as ibuprofen, during lockdown periods. Although there has been a decrease in calls to report possible drug intoxication in the Algarve region, the profile of calls was very similar. Paracetamol was the drug with the highest number of reported cases and the group of psychotropic drugs showed the largest increase between 2019 and 2020.

Biological Consequences of Vanadium Effects on Formation of Reactive Oxygen Species and Lipid Peroxidation.

Lipid peroxidation (LPO), a process that affects human health, can be induced by exposure to vanadium salts and compounds. LPO is often exacerbated by oxidation stress, with some forms of vanadium providing protective effects. The LPO reaction involves the oxidation of the alkene bonds, primarily in polyunsaturated fatty acids, in a chain reaction to form radical and reactive oxygen species (ROS). LPO reactions typically affect cellular membranes through direct effects on membrane structure and function as well as impacting other cellular functions due to increases in ROS. Although LPO effects on mitochondrial function have been studied in detail, other cellular components and organelles are affected. Because vanadium salts and complexes can induce ROS formation both directly and indirectly, the study of LPO arising from increased ROS should include investigations of both processes. This is made more challenging by the range of vanadium species that exist under physiological conditions and the diverse effects of these species. Thus, complex vanadium chemistry requires speciation studies of vanadium to evaluate the direct and indirect effects of the various species that are present during vanadium exposure. Undoubtedly, speciation is important in assessing how vanadium exerts effects in biological systems and is likely the underlying cause for some of the beneficial effects reported in cancerous, diabetic, neurodegenerative conditions and other diseased tissues impacted by LPO processes. Speciation of vanadium, together with investigations of ROS and LPO, should be considered in future biological studies evaluating vanadium effects on the formation of ROS and on LPO in cells, tissues, and organisms as discussed in this review.

FOXO1 represses PPARα-Mediated induction of FGF21 gene expression.

Fibroblast growth factor 21 (FGF21) has emerged as a metabolic regulator that exerts potent anti-diabetic and lipid-lowering effects in animal models of obesity and type 2 diabetes, showing a protective role in fatty liver disease and hepatocellular carcinoma progression. Hepatic expression of FGF21 is regulated by PPARα and is induced by fasting. Ablation of FoxO1 in liver has been shown to increase FGF21 expression in hyperglycemia. To better understand the role of FOXO1 in the regulation of FGF21 expression we have modified HepG2 human hepatoma cells to overexpress FoxO1 and PPARα. Here we show that FoxO1 represses PPARα-mediated FGF21 induction, and that the repression acts on the FGF21 gene promoter without affecting other PPARα target genes. Additionally, we demonstrate that FoxO1 physically interacts with PPARα and that FoxO1/3/4 depletion in skeletal muscle contributes to increased Fgf21 tissue levels. Taken together, these data indicate that FOXO1 is a PPARα-interacting protein that antagonizes PPARα activity on the FGF21 promoter. Because other PPARα target genes remained unaffected, these results suggest a highly specific mechanism implicated in FGF21 regulation. We conclude that FGF21 can be specifically modulated by FOXO1 in a PPARα-dependent manner.

Repurposing Therapeutic Drugs Complexed to Vanadium in Cancer.

Repurposing drugs by uncovering new indications for approved drugs accelerates the process of establishing new treatments and reduces the high costs of drug discovery and development. Metal complexes with clinically approved drugs allow further opportunities in cancer therapy-many vanadium compounds have previously shown antitumor effects, which makes vanadium a suitable metal to complex with therapeutic drugs, potentially improving their efficacy in cancer treatment. In this review, covering the last 25 years of research in the field, we identified non-oncology-approved drugs suitable as ligands to obtain different vanadium complexes. Metformin-decavanadate, vanadium-bisphosphonates, vanadyl(IV) complexes with non-steroidal anti-inflammatory drugs, and cetirizine and imidazole-based oxidovanadium(IV) complexes, each has a parent drug known to have different medicinal properties and therapeutic indications, and all showed potential as novel anticancer treatments. Nevertheless, the precise mechanisms of action for these vanadium compounds against cancer are still not fully understood.

Decavanadate and metformin-decavanadate effects in human melanoma cells.

Decavanadate is a polyoxometalate (POMs) that has shown extensive biological activities, including antidiabetic and anticancer activity. Importantly, vanadium-based compounds as well as antidiabetic biguanide drugs, such as metformin, have shown to exert therapeutic effects in melanoma. A combination of these agents, the metformin-decavanadate complex, was also recognized for its antidiabetic effects and recently described as a better treatment than the monotherapy with metformin enabling lower dosage in rodent models of diabetes. Herein, we compare the effects of decavanadate and metformin-decavanadate on Ca2+-ATPase activity in sarcoplasmic reticulum vesicles from rabbit skeletal muscles and on cell signaling events and viability in human melanoma cells. We show that unlike the decavanadate-mediated non-competitive mechanism, metformin-decavanadate inhibits Ca2+-ATPase by a mixed-type competitive-non-competitive inhibition with an IC50 value about 6 times higher (87 µM) than the previously described for decavanadate (15 µM). We also found that both decavanadate and metformin-decavanadate exert antiproliferative effects on melanoma cells at 10 times lower concentrations than monomeric vanadate. Western blot analysis revealed that both, decavanadate and metformin-decavanadate increased phosphorylation of extracellular signal-regulated kinase (ERK) and serine/threonine protein kinase AKT signaling proteins upon 24 h drug exposure, suggesting that the anti-proliferative activities of these compounds act independent of growth-factor signaling pathways.

Tribbles Pseudokinases in Colorectal Cancer.

The Tribbles family of pseudokinases controls a wide number of processes during cancer on-set and progression. However, the exact contribution of each of the three family members is still to be defined. Their function appears to be context-dependent as they can act as oncogenes or tumor suppressor genes. They act as scaffolds modulating the activity of several signaling pathways involved in different cellular processes. In this review, we discuss the state-of-knowledge for TRIB1, TRIB2 and TRIB3 in the development and progression of colorectal cancer. We take a perspective look at the role of Tribbles proteins as potential biomarkers and therapeutic targets. Specifically, we chronologically systematized all available articles since 2003 until 2020, for which Tribbles were associated with colorectal cancer human samples or cell lines. Herein, we discuss: (1) Tribbles amplification and overexpression; (2) the clinical significance of Tribbles overexpression; (3) upstream Tribbles gene and protein expression regulation; (4) Tribbles pharmacological modulation; (5) genetic modulation of Tribbles; and (6) downstream mechanisms regulated by Tribbles; establishing a comprehensive timeline, essential to better consolidate the current knowledge of Tribbles' role in colorectal cancer.

Harmine and Piperlongumine Revert TRIB2-Mediated Drug Resistance.

Therapy resistance is responsible for most relapses in patients with cancer and is the major challenge to improving the clinical outcome. The pseudokinase Tribbles homologue 2 (TRIB2) has been characterized as an important driver of resistance to several anti-cancer drugs, including the dual ATP-competitive PI3K and mTOR inhibitor dactolisib (BEZ235). TRIB2 promotes AKT activity, leading to the inactivation of FOXO transcription factors, which are known to mediate the cell response to antitumor drugs. To characterize the downstream events of TRIB2 activity, we analyzed the gene expression profiles of isogenic cell lines with different TRIB2 statuses by RNA sequencing. Using a connectivity map-based computational approach, we identified drug-induced gene-expression profiles that invert the TRIB2-associated expression profile. In particular, the natural alkaloids harmine and piperlongumine not only produced inverse gene expression profiles but also synergistically increased BEZ235-induced cell toxicity. Importantly, both agents promote FOXO nuclear translocation without interfering with the nuclear export machinery and induce the transcription of FOXO target genes. Our results highlight the great potential of this approach for drug repurposing and suggest that harmine and piperlongumine or similar compounds might be useful in the clinic to overcome TRIB2-mediated therapy resistance in cancer patients.

Skeletal muscle expression of adipose-specific phospholipase in peripheral artery disease.

Flow-limiting atherosclerotic lesions of arteries supplying the limbs are a cause of symptoms in patients with peripheral artery disease (PAD). Musculoskeletal metabolic factors also contribute to the pathophysiology of claudication, which is manifest as leg discomfort that impairs walking capacity. Accordingly, we conducted a case-control study to determine whether skeletal muscle metabolic gene expression is altered in PAD. Calf skeletal muscle gene expression of patients with PAD and healthy subjects was analyzed using microarrays. The top-ranking gene differentially expressed between PAD and controls (FDR < 0.001) was PLA2G16, which encodes adipose-specific phospholipase A2 (AdPLA) and is implicated in the maintenance of insulin sensitivity and regulation of lipid metabolism. Differential expression was confirmed by qRT-PCR; PLA2G16 was downregulated by 68% in patients with PAD (p < 0.001). Expression of Pla2g16 was then measured in control (db/+) and diabetic (db/db) mice that underwent unilateral femoral artery ligation. There was significantly reduced expression of Pla2g16 in the ischemic leg of both control and diabetic mice (by 51%), with significantly greater magnitude of reduction in the diabetic mice (by 79%). We conclude that AdPLA is downregulated in humans with PAD and in mice with hindlimb ischemia. Reduced AdPLA may contribute to impaired walking capacity in patients with PAD via its effects on skeletal muscle metabolism. Further studies are needed to fully characterize the role of AdPLA in PAD and to investigate its potential as a therapeutic target for alleviating symptoms of claudication.

Health literacy assessment: Translation and cultural adaptation to the Portuguese population.

RATIONALE, AIMS, AND OBJECTIVES: Health literacy (HL) has been widely referenced as a determinant of health outcomes, making the assessment of low HL a fundamental step to plan educational interventions. This study aimed to translate and adapt the Short Assessment of Health Literacy-Spanish and English (SAHL-S&E) questionnaire into European Portuguese. METHODS: The SAHL-S&E questionnaire was translated using the recommendations of the International Society for Pharmacoeconomics and Outcomes Research. One hundred fifty-three Portuguese native speakers aged over 18 years old were included in this study, enrolled among users of community pharmacies in the Algarve region (Portugal). RESULTS: The translation of the questionnaire used showed a good internal consistency (Cronbach α: .812), and a statistically significant (F = 5.05 P < .001) interrater reliability. Over a third of subjects (37.9%) achieved a score less than or equal to 14, which is indicative of low HL. CONCLUSION: This tool, intended to be used in the European Portuguese population, can be used for low HL screening.

Paternal Exercise Improves Glucose Metabolism in Adult Offspring.

Poor paternal diet has emerged as a risk factor for metabolic disease in offspring, and alterations in sperm may be a major mechanism mediating these detrimental effects of diet. Although exercise in the general population is known to improve health, the effects of paternal exercise on sperm and offspring metabolic health are largely unknown. Here, we studied 7-week-old C57BL/6 male mice fed a chow or high-fat diet and housed either in static cages (sedentary) or cages with attached running wheels (exercise trained). After 3 weeks, one cohort of males was sacrificed and cauda sperm obtained, while the other cohort was bred with chow-fed sedentary C57BL/6 females. Offspring were chow fed, sedentary, and studied during the first year of life. We found that high-fat feeding of sires impairs glucose tolerance and increases the percentage of fat mass in both male and female offspring at 52 weeks of age. Strikingly, paternal exercise suppresses the effects of paternal high-fat diet on offspring, reversing the observed impairment in glucose tolerance, percentage of fat mass, and glucose uptake in skeletal muscles of the offspring. These changes in offspring phenotype are accompanied by changes in sperm physiology, as, for example, high-fat feeding results in decreased sperm motility, an effect normalized in males subject to exercise training. Deep sequencing of sperm reveals pronounced effects of exercise training on multiple classes of small RNAs, as multiple changes to the sperm RNA payload observed in animals consuming a high-fat diet are suppressed by exercise training. Thus, voluntary exercise training of male mice results in pronounced improvements in the metabolic health of adult male and female offspring. We provide the first in-depth analysis of small RNAs in sperm from exercise-trained males, revealing a marked change in the levels of multiple small RNAs with the potential to alter phenotypes in the next generation.

l-Alanine activates hepatic AMP-activated protein kinase and modulates systemic glucose metabolism.

OBJECTIVE: AMP activated protein kinase (AMPK) is recognized as an important nutrient sensor contributing to regulation of cellular, tissue, and systemic metabolism. We aimed to identify specific amino acids which could modulate AMPK and determine effects on cellular and systemic metabolism. METHODS: We performed an unbiased amino acid screen to identify activators of AMPK. Detailed analysis of cellular signaling and metabolism was performed in cultured hepatoma cells, and in vivo glucose metabolism and metabolomic patterns were assessed in both chow-fed mice and mice made obese by high-fat diet feeding. RESULTS: Alanine acutely activates AMP kinase in both cultured hepatic cells and in liver from mice treated in vivo with Ala. Oral alanine administration improves systemic glucose tolerance in both chow and high fat diet fed mice, with reduced efficacy of Ala in mice with reduced AMPK activity. Our data indicate that Ala activation of AMPK is mediated by intracellular Ala metabolism, which reduces TCA cycle metabolites, increases AMP/ATP ratio, and activates NH3 generation. CONCLUSIONS: Ala may serve as a distinct amino acid energy sensor, providing a positive signal to activate the beneficial AMPK signaling pathway.

Defects in muscle branched-chain amino acid oxidation contribute to impaired lipid metabolism.

OBJECTIVE: Plasma levels of branched-chain amino acids (BCAA) are consistently elevated in obesity and type 2 diabetes (T2D) and can also prospectively predict T2D. However, the role of BCAA in the pathogenesis of insulin resistance and T2D remains unclear. METHODS: To identify pathways related to insulin resistance, we performed comprehensive gene expression and metabolomics analyses in skeletal muscle from 41 humans with normal glucose tolerance and 11 with T2D across a range of insulin sensitivity (SI, 0.49 to 14.28). We studied both cultured cells and mice heterozygous for the BCAA enzyme methylmalonyl-CoA mutase (Mut) and assessed the effects of altered BCAA flux on lipid and glucose homeostasis. RESULTS: Our data demonstrate perturbed BCAA metabolism and fatty acid oxidation in muscle from insulin resistant humans. Experimental alterations in BCAA flux in cultured cells similarly modulate fatty acid oxidation. Mut heterozygosity in mice alters muscle lipid metabolism in vivo, resulting in increased muscle triglyceride accumulation, increased plasma glucose, hyperinsulinemia, and increased body weight after high-fat feeding. CONCLUSIONS: Our data indicate that impaired muscle BCAA catabolism may contribute to the development of insulin resistance by perturbing both amino acid and fatty acid metabolism and suggest that targeting BCAA metabolism may hold promise for prevention or treatment of T2D.

Metabolic Effects of Long-Term Reduction in Free Fatty Acids With Acipimox in Obesity: A Randomized Trial.

CONTEXT: Increased circulating free fatty acids (FFAs) have been proposed to contribute to insulin resistance in obesity. Short-term studies have investigated the effects of acipimox, an inhibitor of hormone-sensitive lipase, on glucose homeostasis, but longer-term studies have not been performed. OBJECTIVE: To test the hypothesis that long-term treatment with acipimox would reduce FFA and improve insulin sensitivity among nondiabetic, insulin-resistant, obese subjects. DESIGN, SETTING, PATIENTS, AND INTERVENTION: At an academic medical center, 39 obese men and women were randomized to acipimox 250 mg thrice-daily vs identical placebo for 6 months. MAIN OUTCOME MEASURES: Plasma lipids, insulin sensitivity, adiponectin, and mitochondrial function via assessment of the rate of post-exercise phosphocreatine recovery on (31)P-magnetic resonance spectroscopy as well as muscle mitochondrial density and relevant muscle gene expression. RESULTS: Fasting glucose decreased significantly in acipimox-treated individuals (effect size, -6 mg/dL; P = .02), in parallel with trends for reduced fasting insulin (effect size, -6.8 µU/mL; P = .07) and HOMA-IR (effect size, -1.96; P = .06), and significantly increased adiponectin (effect size, +668 ng/mL; P = .02). Acipimox did not affect insulin-stimulated glucose uptake, as assessed by euglycemic, hyperinsulinemic clamp. Effects on muscle mitochondrial function and density and on relevant gene expression were not seen. CONCLUSION: These data shed light on the long-term effects of FFA reduction on insulin sensitivity, other metabolic parameters, and muscle mitochondrial function in obesity. Reduced FFA achieved by acipimox improved fasting measures of glucose homeostasis, lipids, and adiponectin but had no effect on mitochondrial function, mitochondrial density, or muscle insulin sensitivity.

FGF21 mediates the lipid metabolism response to amino acid starvation.

Lipogenic gene expression in liver is repressed in mice upon leucine deprivation. The hormone fibroblast growth factor 21 (FGF21), which is critical to the adaptive metabolic response to starvation, is also induced under amino acid deprivation. Upon leucine deprivation, we found that FGF21 is needed to repress expression of lipogenic genes in liver and white adipose tissue, and stimulate phosphorylation of hormone-sensitive lipase in white adipose tissue. The increased expression of Ucp1 in brown adipose tissue under these circumstances is also impaired in FGF21-deficient mice. Our results demonstrate the important role of FGF21 in the regulation of lipid metabolism during amino acid starvation.

Fsp27/CIDEC is a CREB target gene induced during early fasting in liver and regulated by FA oxidation rate.

FSP27 [cell death-inducing DFFA-like effector c (CIDEC) in humans] is a protein associated with lipid droplets that downregulates the fatty acid oxidation (FAO) rate when it is overexpressed. However, little is known about its physiological role in liver. Here, we show that fasting regulates liver expression of Fsp27 in a time-dependent manner. Thus, during the initial stages of fasting, a maximal induction of 800-fold was achieved, whereas during the later phase of fasting, Fsp27 expression decreased. The early response to fasting can be explained by a canonical PKA-CREB-CRTC2 signaling pathway because: i) CIDEC expression was induced by forskolin, ii) Fsp27 promoter activity was increased by CREB, and iii) Fsp27 expression was upregulated in the liver of Sirt1 knockout animals. Interestingly, pharmacological (etomoxir) or genetic (Hmgcs2 interference) inhibition of the FAO rate increases the in vivo expression of Fsp27 during fasting. Similarly, CIDEC expression was upregulated in HepG2 cells by either etomoxir or HMGCS2 interference. Our data indicate that there is a kinetic mechanism of autoregulation between short- and long-term fasting, by which free FAs delivered to the liver during early fasting are accumulated/exported by FSP27/CIDEC, whereas over longer periods of fasting, they are degraded in the mitochondria through the carnitine palmitoyl transferase system.

Activating transcription factor 4-dependent induction of FGF21 during amino acid deprivation.

Nutrient deprivation or starvation frequently correlates with amino acid limitation. Amino acid starvation initiates a signal transduction cascade starting with the activation of the kinase GCN2 (general control non-derepressible 2) phosphorylation of eIF2 (eukaryotic initiation factor 2), global protein synthesis reduction and increased ATF4 (activating transcription factor 4). ATF4 modulates a wide spectrum of genes involved in the adaptation to dietary stress. The hormone FGF21 (fibroblast growth factor 21) is induced during fasting in liver and its expression induces a metabolic state that mimics long-term fasting. Thus FGF21 is critical for the induction of hepatic fat oxidation, ketogenesis and gluconeogenesis, metabolic processes which are essential for the adaptive metabolic response to starvation. In the present study, we have shown that FGF21 is induced by amino acid deprivation in both mouse liver and cultured HepG2 cells. We have identified the human FGF21 gene as a target gene for ATF4 and we have localized two conserved ATF4-binding sequences in the 5' regulatory region of the human FGF21 gene, which are responsible for the ATF4-dependent transcriptional activation of this gene. These results add FGF21 gene induction to the transcriptional programme initiated by increased levels of ATF4 and offer a new mechanism for the induction of the FGF21 gene expression under nutrient deprivation.

Human HMGCS2 regulates mitochondrial fatty acid oxidation and FGF21 expression in HepG2 cell line.

HMGCS2 (hydroxymethylglutaryl CoA synthase 2), the gene that regulates ketone body production, is barely expressed in cultured cell lines. In this study, we restored HMGCS2 expression and activity in HepG2 cells, thus showing that the wild type enzyme can induce fatty acid ß-oxidation (FAO) and ketogenesis, whereas a catalytically inactive mutant C166A did not generate either process. Peroxisome proliferator-activated receptor (PPAR) α expression also induces fatty acid ß-oxidation and endogenous HMGCS2 expression. Interestingly, PPARα-mediated induction was abolished when HMGCS2 expression was down-regulated by RNAi. These results indicate that HMGCS2 expression is both sufficient and necessary to the control of fatty acid oxidation in these cells. Next, we examined the expression pattern of several PPARα target genes in this now "ketogenic" HepG2 cell line. FGF21 (fibroblast growth factor 21) expression was specifically induced by HMGCS2 activity or by the inclusion of the oxidized form of ketone bodies (acetoacetate) in the culture medium. This effect was blunted by SirT1 (sirtuin 1) RNAi, so we propose a SirT1-dependent mechanism for FGF21 induction by acetoacetate. These data suggest a novel feed-forward mechanism by which HMGCS2 could regulate adaptive metabolic responses during fasting. This mechanism could be physiologically relevant, because fasting-mediated induction of liver FGF21 was dependent on SirT1 activity in vivo.