Intraarticular monomethyl fumarate as a perspective therapy for osteoarthritis by macrophage polarization.

BACKGROUND: Osteoarthritis (OA) is a chronic disease that may lead to joint structure degeneration, cartilage destruction, osteophyte formation, subchondral bone disruption, and pain. In this scenario, a higher proportion of the proinflammatory macrophage type 1 (M1) than the anti-inflammatory macrophage type 2 (M2) could be highlighted as a hallmark of OA progression. The balance between these two macrophage types emerges as a new therapeutic target in OA. This study aimed to evaluate the analgesia and macrophage profile in the treatment of experimental osteoarthritis (EOA) with systemic dimethyl fumarate (DMF) or local intra-articular monomethyl fumarate (MMF). RESULTS: DMF via gavage or MMF via intra-articular in the right knee of EOA rats showed improvements in gait parameters and the nociceptive recovery of the mechanical threshold assessment by adapted electronic von Frey treatment on the twenty-first day (long-lasting phase). DMF treatment decreased proinflammatory TNF-α while increasing anti-inflammatory IL-10 cytokines from the macerated capsule on the fifth day (inflammatory phase). MMF treatment showed joint capsule mRNA extraction downregulating iNOS and TNF-α gene expression while upregulating IL-10 and MCP-1. However, CD206 was not significant but higher than untreated EOA rats' joints on the seventh day (inflammatory phase). CONCLUSIONS: Our studies with EOA model induced by MIA suggest a new perspective for human treatment committed with OA based on macrophage polarization as a therapeutic target, switching the proinflammatory profile M1 to the anti-inflammatory profile M2 with DMF systematic or by MMF locally treatment according to the OA severity.

Agomelatine inhibits platelet aggregation through melatonin receptor-dependent and independent mechanisms.

AIMS: Melatonin is known to inhibit platelet aggregation induced by arachidonic acid (AA). In the present study we investigated whether agomelatine (Ago), an antidepressant with agonist activity at melatonin receptor 1 (MT1) and MT2 could reduce platelets aggregation and adhesion. MAIN METHODS: Human platelets from healthy donors were used to test the in vitro effects of Ago in the presence of different platelet activators. We performed aggregation and adhesion assays, thromboxane B2 (TxB2), cAMP and cGMP measurements, intra-platelet calcium registration and flow cytometry assays. KEY FINDINGS: Our data revealed that different concentrations of Ago reduced AA- and collagen-induced human platelet aggregation in vitro. Ago also reduced AA-induced increase in thromboxane B2 (TxB2) production, intracellular calcium levels and P-selectin expression at plasma membrane. The effects of Ago in AA-activated platelets were likely dependent on MT1 as they were blocked by luzindole (a MT1/MT2 antagonist) and mimicked by the MT1 agonist UCM871 in a luzindole-sensitive manner. The MT2 agonist UCM924 was also able to inhibit platelet aggregation, but this response was not affected by luzindole. On the other hand, although UCM871 and UCM924 reduced collagen-induced platelet aggregation and adhesion, inhibition of collagen-induced platelet aggregation by Ago was not mediated by melatonin receptors because it was not affected by luzindole. SIGNIFICANCE: The present data show that Ago suppresses human platelet aggregation and suggest that this antidepressant may have the potential to prevent atherothrombotic ischemic events by reducing thrombus formation and vessel occlusion.

Effectiveness of Oral Hydration in Preventing Contrast-Induced Nephropathy in Individuals Undergoing Elective Coronary Interventions. / Eficácia da Hidratação Oral na Prevenção da Nefropatia Induzida por Contraste em Indivíduos Submetidos a Intervenções Coronárias Eletivas.

BACKGROUND: Contrast-induced nephropathy (CIN) is defined as worsening renal function, represented by an increase in serum creatinine of ≥ 25% or ≥ 0.5 mg/dL up to 72 h after exposure to iodinated contrast medium (ICM). The most effective preventive measure to date is intravenous hydration (IVH). Little is known about the effectiveness of outpatient oral hydration (OH). OBJETIVE: To investigate whether outpatient OH with water is as effective as IVH with 0.9% saline solution in preventing CIN in elective coronary procedures. METHODS: In this retrospective observational study, we analyzed the medical records and laboratory data of individuals undergoing percutaneous coronary procedures with ICM. Data collected between 2012 and 2015 refer to individuals who underwent IVH and those collected between 2016 and 2020 (after implementation of an OH protocol) correspond to individuals who underwent OH at home before and after coronary procedures as instructed by the nursing team. Statistical significance was established at α = 0.05. RESULTS: In total, 116 patients were included in this study: 58 in the IVH group and 58 in the OH group. An incidence of CIN of 15% (9/58) was observed in the group that received IVH and an incidence of 12% (7/58) was seen in the group that received OH (p = 0.68). CONCLUSION: The OH protocol, performed by the patient, appears to be as effective as the in-hospital IVH protocol for the renal protection of individuals susceptible to CIN in elective coronary interventions. These findings should be put to test in larger trials.

FUNDAMENTO: A nefropatia induzida por contraste (NIC) é definida como deterioração da função renal, representada por um aumento da creatinina sérica ≥25% ou ≥0,5 mg/dL até 72 horas após a exposição ao meio de contraste iodado (MCI). A medida preventiva mais eficaz até o momento é a hidratação venosa (HV). Pouco se sabe sobre a eficácia da hidratação oral (HO) ambulatorial. OBJETIVO: Investigar se a HO ambulatorial com água é tão eficaz quanto a HV com solução salina a 0,9% na prevenção de NIC em procedimentos coronarianos eletivos. MÉTODOS: Neste estudo observacional retrospectivo, foram analisados prontuários médicos e dados laboratoriais para coletar dados de indivíduos submetidos a procedimentos coronarianos percutâneos com MCI. Os dados coletados entre 2012 e 2015 avaliaram indivíduos que foram submetidos à HV e entre 2016 e 2020 (após a implementação de um protocolo de HO), os indivíduos que foram submetidos à HO em casa antes e depois de procedimentos coronarianos, conforme orientação da equipe de enfermagem. A significância estatística adotada foi de α=0,05. RESULTADOS: No total, 116 pacientes foram incluídos neste estudo, 58 no grupo HV e 58 no grupo HO. Observou-se incidência de NIC de 15% (9/58) no grupo que recebeu HV e 12% (7/58) no grupo que recebeu HO (p=0,68). CONCLUSÃO: O protocolo de HO realizado pelo paciente parece ser tão eficaz quanto o protocolo de HV hospitalar na proteção renal de indivíduos suscetíveis a desenvolver NIC em intervenções coronarianas eletivas. Essas descobertas devem ser testadas em ensaios mais abrangentes.

Eficácia da Hidratação Oral na Prevenção da Nefropatia Induzida por Contraste em Indivíduos Submetidos a Intervenções Coronárias Eletivas / Effectiveness of Oral Hydration in Preventing Contrast-Induced Nephropathy in Individuals Undergoing Elective Coronary Interventions

Resumo Fundamento A nefropatia induzida por contraste (NIC) é definida como deterioração da função renal, representada por um aumento da creatinina sérica ≥25% ou ≥0,5 mg/dL até 72 horas após a exposição ao meio de contraste iodado (MCI). A medida preventiva mais eficaz até o momento é a hidratação venosa (HV). Pouco se sabe sobre a eficácia da hidratação oral (HO) ambulatorial. Objetivo Investigar se a HO ambulatorial com água é tão eficaz quanto a HV com solução salina a 0,9% na prevenção de NIC em procedimentos coronarianos eletivos. Métodos Neste estudo observacional retrospectivo, foram analisados prontuários médicos e dados laboratoriais para coletar dados de indivíduos submetidos a procedimentos coronarianos percutâneos com MCI. Os dados coletados entre 2012 e 2015 avaliaram indivíduos que foram submetidos à HV e entre 2016 e 2020 (após a implementação de um protocolo de HO), os indivíduos que foram submetidos à HO em casa antes e depois de procedimentos coronarianos, conforme orientação da equipe de enfermagem. A significância estatística adotada foi de α=0,05. Resultados No total, 116 pacientes foram incluídos neste estudo, 58 no grupo HV e 58 no grupo HO. Observou-se incidência de NIC de 15% (9/58) no grupo que recebeu HV e 12% (7/58) no grupo que recebeu HO (p=0,68). Conclusão O protocolo de HO realizado pelo paciente parece ser tão eficaz quanto o protocolo de HV hospitalar na proteção renal de indivíduos suscetíveis a desenvolver NIC em intervenções coronarianas eletivas. Essas descobertas devem ser testadas em ensaios mais abrangentes.

Abstract Background Contrast-induced nephropathy (CIN) is defined as worsening renal function, represented by an increase in serum creatinine of ≥ 25% or ≥ 0.5 mg/dL up to 72 h after exposure to iodinated contrast medium (ICM). The most effective preventive measure to date is intravenous hydration (IVH). Little is known about the effectiveness of outpatient oral hydration (OH). Objetive To investigate whether outpatient OH with water is as effective as IVH with 0.9% saline solution in preventing CIN in elective coronary procedures. Methods In this retrospective observational study, we analyzed the medical records and laboratory data of individuals undergoing percutaneous coronary procedures with ICM. Data collected between 2012 and 2015 refer to individuals who underwent IVH and those collected between 2016 and 2020 (after implementation of an OH protocol) correspond to individuals who underwent OH at home before and after coronary procedures as instructed by the nursing team. Statistical significance was established at α = 0.05. Results In total, 116 patients were included in this study: 58 in the IVH group and 58 in the OH group. An incidence of CIN of 15% (9/58) was observed in the group that received IVH and an incidence of 12% (7/58) was seen in the group that received OH (p = 0.68). Conclusion The OH protocol, performed by the patient, appears to be as effective as the in-hospital IVH protocol for the renal protection of individuals susceptible to CIN in elective coronary interventions. These findings should be put to test in larger trials.

Low Birth Weight Intensifies Changes in Markers of Hepatocarcinogenesis Induced by Fructose Consumption in Rats.

Intrauterine growth restriction (IUGR) due to fetal exposure to glucocorticoid excess results in metabolic inflexibility and hepatic steatosis upon nutritional stress during adulthood. We previously demonstrated that rats born to dexamethasone (DEX)-treated mothers developed hepatic steatosis when exposed to 10% fructose solution during adult life. Persistent triacylglyceride (TAG) accumulation in the liver, in turn, is a feature of non-alcoholic fatty liver disease (NAFLD), which serves as a risk factor for non-alcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC). In the present study, we demonstrate that the combination of IUGR and fructose treatment during adulthood also results in increased hepatic myeloperoxidase (MPO) activity, AKT phosphorylation and serum aspartate transaminase. Growth-restricted rats also presented reduced hepatic TRIB3 and GADD45a after fructose treatment. Other markers of cell proliferation, such as Cyclin D, PCNA, Hgf and Hspa4/Hsp70 expression and the number of Ki-67 positive cells, were all increased in the liver of growth- restricted rats treated with fructose. On the other hand, the combination of IUGR and fructose treatment during adult life reduced the levels of IGF-1. In conclusion, our data indicate that after exposure to fructose, adult rats subjected to dexamethasone-induced IUGR display exacerbated molecular changes in markers of NASH and HCC.

Antenatal corticosteroid therapy modulates hepatic AMPK phosphorylation and maternal lipid metabolism in early lactating rats.

Antenatal corticosteroid therapy is used to reduce neonatal mortality in preterm infants but it is currently unknown whether this intervention affects lipid metabolism at the peripartum. This study aimed to evaluate if antenatal corticosteroid therapy in pregnant rats and women affects lipid metabolism during early lactation. We evaluated women at risk of preterm delivery that received corticosteroid therapy (CASE) and women that were not exposed to corticosteroid and were not at risk of preterm delivery (CONTROL). Samples were collected to measure serum and milk triacylglycerol (TAG) three days after delivery. Rats were treated with dexamethasone (DEX) between the 15th and the 20th days of pregnancy. Samples were collected at different days after delivery (L3, L8 and L14). TAG was measured in serum, liver and mammary gland (MG). TAG appearance rates were measured after tyloxapol injection and gavage with olive oil. We also evaluated the expression of key genes related to lipid metabolism in the liver and in the MG and hepatic phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC). CASE volunteers delivered earlier than CONTROL but presented unaltered milk and serum TAG concentrations. Early lactating DEX rats exhibited increased TAG in serum, MG and milk. No changes in CD36 and LPL were detected in the MG and liver. Early lactating DEX rats displayed increased TAG appearance rate and reduced hepatic AMPK/ACC phosphorylation. Our data revealed that antenatal corticosteroid therapy reduces hepatic AMPK/ACC phosphorylation during early lactation that reflects in increased TAG concentration in serum, MG and milk.

Agomelatine reduces circulating triacylglycerides and hepatic steatosis in fructose-treated rats.

Agomelatine (AGO) is an antidepressant drug with agonistic activity at melatonin receptor 1 (MT1) and MT2 and with neutral antagonistic activity at serotonin receptor 5-HT2C. Although experimental studies show that melatonin reduces hypertriglyceridemia and hepatic steatosis induced by excessive fructose intake, no studies have tested if AGO exerts similar actions. To address this issue we have treated male Wistar rats with fructose (15% in the drinking water) and/or AGO (40 mg/kg/day) for two weeks. AGO reduced body weight gain, feeding efficiency and hepatic lipid levels without affecting caloric intake in fructose-treated rats. AGO has also decreased very low-density lipoprotein (VLDL) production and circulating TAG levels after an oral load with olive oil. Accordingly, treatment with AGO reduced the hepatic expression of fatty acid synthase (Fasn), a limiting step for hepatic de novo lipogenesis (DNLG). The expression of apolipoprotein B (Apob) and microsomal triglyceride transfer protein (Mttp) in the ileum, two crucial proteins for intestinal lipoprotein production, were also downregulated by treatment with AGO. Altogether, the present data show that AGO mimics the metabolic benefits of melatonin when used in fructose-treated rats. This study also suggests that it is relevant to evaluate the potential of AGO to treat metabolic disorders in future clinical trials.

Dexamethasone programs lower fatty acid absorption and reduced PPAR-γ and fat/CD36 expression in the jejunum of the adult rat offspring.

The progeny of rats born and breastfed by mothers receiving dexamethasone (DEX) during pregnancy exhibits permanent reduction in body weight and adiposity but the precise mechanisms related to this programming are not fully understood. In order to clarify this issue, the present study investigated key aspects of lipoprotein production and lipid metabolism by the liver and the intestine that would explain the reduced adiposity seen in the adult offspring exposed to DEX in utero. Female Wistar rats were treated with DEX (0.1 mg/kg/day) between the 15th and the 21st days of pregnancy, while control mothers were treated with vehicle. Male offspring born to control mothers were nursed by either adoptive control mothers (CTL/CTL) or DEX-treated mothers (CTL/DEX). Male offspring born to DEX-treated mothers were nursed by either control mothers (DEX/CTL) or adoptive DEX-treated mothers (DEX/DEX). We found that only the male DEX/DEX offspring had reduced adiposity. Additionally, male DEX/DEX progeny had lower circulating triacylglycerol (TAG) levels only in fed-state. The four groups of offspring presented similar energy expenditure, respiratory quotient and very low-density lipoprotein (VLDL) production. On the other hand, DEX/DEX rats displayed reduced TAG levels after gavage with olive oil and reduced expression of fatty acid translocase Cd36 (Fat/Cd36) and peroxisome proliferator-activated receptor γ (Pparg) in the jejunum. Altogether, our study supports the notion that reduced fat absorption by the jejunum may contribute to the lower adiposity of the adult offspring born and breastfed by mothers treated with DEX during pregnancy.

The sodium-glucose cotransporter-2 (SGLT2) inhibitors synergize with nitric oxide and prostacyclin to reduce human platelet activation.

Gliflozins (canagliflozin, dapagliflozin and empagliflozin) are the newest anti-hyperglycemic class and have offered cardiovascular and renal benefits. Because platelets are involved in the atherothrombosis process, this study is aimed to evaluate the direct effect of gliflozins on platelet reactivity. Platelet-rich plasma (PRP) or washed platelets (WP) were obtained from healthy volunteers. Aggregation, flow cytometry for glycoprotein IIb/IIIa, cyclic nucleotides and intracellular calcium levels, Western blot, thromboxane B2 (TXB2) measurement and COX-1 activity were performed in the presence of gliflozins (1-30 µM) alone or in combination with sodium nitroprusside (SNP, 10 or 100 nM) + iloprost (ILO, 0.1 or 1 nM). SGLT2 protein is not expressed on human platelets. Gliflozins produced little inhibitory effect in agonists-induced aggregation and this effect was greatly potentiated by ~10-fold in the presence of SNP + ILO, accompanied by lower levels of TXB2 (58.1 ± 5.1%, 47.1 ± 7.2% and 43.4 ± 9.2% inhibition for canagliflozin, dapagliflozin and empagliflozin, respectively). The activity of COX-1 was not affected by gliflozins. Collagen increased Ca2+ levels and α(IIb)ß(3) activation, both of which were significantly reduced by gliflozins + SNP + ILO. The intracellular levels of cAMP and cGMP and the protein expression of p-VASPSer157 and p-VASPSer239 were not increased by gliflozins while the expression of the serine-threonine kinase, AktSer473 was markedly reduced. Our results showed that the antiplatelet activity of gliflozins were greatly enhanced by nitric oxide and prostacyclin, thus suggesting that the cardiovascular protection seen by this class of drugs could be in part due to platelet inhibition.

In utero exposure to dexamethasone programs the development of the pancreatic ß- and α-cells during early postnatal life.

AIMS: The aim of the present study was to clarify if in utero exposure to DEX would affect the development of different types of pancreatic endocrine cells during postnatal life. MAIN METHODS: We investigated morphological and transcriptional features of both pancreatic ß- and α-cell populations within the pancreatic islets during the early postnatal life of rats born to mothers treated with DEX (0.1 mg/kg) from day 14 to 19 of pregnancy. Untreated pregnant Wistar rats of the same age (12-week-old) were used as control (CTL). Pups were euthanized on the 1st, 3rd and 21st (PND1, PND3 and PND21, respectively) days of life, regardless of sex. Serum insulin and glucagon levels were also evaluated. KEY FINDINGS: Rats born to DEX-treated mothers exhibited increased pancreatic α-cell mass, circulating glucagon levels and Gcg, Pax6, MafB and Nkx2.2 expression. Rats born to DEX-treated mothers also presented a rise in serum insulin levels on the PND3 that was paralleled by reduced ß-cell mass. Such increase in serum insulin levels, instead, was associated with increased expression of genes associated to insulin secretion such as Gck and Slc2a2. SIGNIFICANCE: Altogether, the present data reveals yet unknown changes in endocrine pancreas during early postnatal life of rats exposed to DEX in utero. Such data may contribute to the understanding of the metabolic features of rats born to DEX-treated mothers.

Long-term increase of insulin secretion in mice subjected to pregnancy and lactation.

PURPOSE: Observational studies show that longer breastfeeding periods reduce maternal risk of type 2 diabetes mellitus. However, it is currently unknown if the long-term benefits of breastfeeding for maternal glucose homeostasis are linked to changes in the endocrine pancreas. METHODS: We presently evaluated functional, morphological and molecular aspects of the endocrine pancreas of mice subjected to two sequential cycles of pregnancy and lactation (L21). Age-matched mice not allowed to breastfeed (L0) and virgin mice were used as controls. RESULTS: L21 mice exhibited increased tolerance and increased glucose-stimulated insulin secretion (GSIS) by isolated islets. Pancreatic islets of L21 mice did not present evident morphological changes to justify the increased GSIS. On the other hand, islets of L21 mice exhibited a reduction in Cavb3 and Kir6.2 expression with concordant increased intracellular Ca2+ levels after challenge with glucose. CONCLUSION: Altogether, the present findings show the breastfeeding exerts long-term benefits for maternal endocrine pancreas by increasing intracellular Ca2+ levels and GSIS.

Maternal Obesity in Mice Exacerbates the Allergic Inflammatory Response in the Airways of Male Offspring.

: It was previously demonstrated that non-allergen-sensitized rodents born to mothers exposed to a high-fat diet (HFD) spontaneously develop lower respiratory compliance and higher respiratory resistance. In the present study, we sought to determine if mice born to mothers consuming HFD would exhibit changes in inflammatory response and lung remodeling when subjected to ovalbumin (OVA) sensitization/challenge in adult life. Mice born to dams consuming either HFD or standard chow had increased bronchoalveolar lavage (BAL) levels of IL-1ß, IL-4, IL-5, IL-10, IL-13, TNF-α and TGF-ß1 after challenge with OVA. IL-4, IL-13, TNF-α and TGF-ß1 levels were further increased in the offspring of HFD-fed mothers. Mice born to obese dams also had exacerbated values of leukocyte infiltration in lung parenchyma, eosinophil and neutrophil counts in BAL, mucus overproduction and collagen deposition. The programming induced by maternal obesity was accompanied by increased expression of miR-155 in peripheral-blood mononuclear cells and reduced miR-133b in trachea and lung tissue in adult life. Altogether, the present data support the unprecedented notion that the progeny of obese mice display exacerbated responses to sensitization/challenge with OVA, leading to the intensification of the morphological changes of lung remodeling. Such changes are likely to result from long-lasting changes in miR-155 and miR-133b expression.

Dexamethasone during pregnancy impairs maternal pancreatic ß-cell renewal during lactation.

Pancreatic islets from pregnant rats develop a transitory increase in the pancreatic ß-cell proliferation rate and mass. Increased apoptosis during early lactation contributes to the rapid reversal of those morphological changes. Exposure to synthetic glucocorticoids during pregnancy has been previously reported to impair insulin secretion, but its impacts on pancreatic islet morphological changes during pregnancy and lactation have not been described. To address this issue, we assessed the morphological and molecular characteristics of pancreatic islets from rats that underwent undisturbed pregnancy (CTL) or were treated with dexamethasone between the 14th and 19th days of pregnancy (DEX). Pancreatic islets were analyzed on the 20th day of pregnancy (P20) and on the 3rd, 8th, 14th and 21st days of lactation (L3, L8, L14 and L21, respectively). Pancreatic islets from CTL rats exhibited transitory increases in cellular proliferation and pancreatic ß-cell mass at P20, which were reversed at L3, when a transitory increase in apoptosis was observed. This was followed by the appearance of morphological features of pancreatic islet neogenesis at L8. Islets from DEX rats did not demonstrate an increase in apoptosis at L3, which coincided with an increase in the expression of M2 macrophage markers relative to M1 macrophage and T lymphocyte markers. Islets from DEX rats also did not exhibit the morphological characteristics of pancreatic islet neogenesis at L8. Our data demonstrate that maternal pancreatic islets undergo a renewal process during lactation that is impaired by exposure to DEX during pregnancy.

The absence of lactation after pregnancy induces long-term lipid accumulation in maternal liver of mice.

AIMS: The present investigation evaluated whether pregnancy followed by lactation exerts long-term impacts on maternal hepatic lipid metabolism. MAIN METHODS: Female mice were subjected to two pregnancies, after which they were either allowed to breastfeed their pups for 21 days (L21) or had their litter removed (L0). Age-matched virgin mice were used as controls (CTL). Three months after the second delivery, serum was collected for lipid profiling, and fragments of liver were used to assess lipid content and to evaluate the key steps of de novo non-esterified fatty acid (NEFA) synthesis, esterification and ß-oxidation, very low density lipoprotein (VLDL) assembly and secretion and autophagy. KEY FINDINGS: L0 exhibited a significant increase in hepatic TG and reduced apolipoprotein B-100 (ApoB-100) expression. L21 mice had increased ATP citrate lyase (ACLY) activity and reduced acetyl-CoA carboxylase (ACC) phosphorylation but no increased hepatic TG. On the other hand, L21 mice had reduced hepatic sequestosome 1 (SQSTM1/p62) levels. Increased high density lipoprotein (HDL) cholesterol and hepatic apolipoprotein A-1 (ApoA-1) expression were found exclusively in L21. SIGNIFICANCE: The present study reveals that long-term hepatic lipid accumulation is induced by the history of pregnancy without lactation. On the other hand, reduced SQSTM1/p62 levels indicate that increased autophagic flux during life may prevent hepatic fat in dams subjected to lactation. Lactation after pregnancy is also obligatory for a long-term increase in maternal HDL. The present data may contribute to the understanding of the mechanisms leading to elevated cardiometabolic risk in women limited to short periods of lactation.

Palmitate-induced Slc2a4/GLUT4 downregulation in L6 muscle cells: evidence of inflammatory and endoplasmic reticulum stress involvement.

BACKGROUND: Obesity is strongly associated to insulin resistance, inflammation, and elevated plasma free fatty acids, but the mechanisms behind this association are not fully comprehended. Evidences suggest that endoplasmic reticulum (ER) stress may play a role in this complex pathophysiology. The aim of the present study was to investigate the involvement of inflammation and ER stress in the modulation of glucose transporter GLUT4, encoded by Slc2a4 gene, in L6 skeletal muscle cells. METHODS: L6 cells were acutely (2 h) and chronically (6 and 12 h) exposed to palmitate, and the expression of several proteins involved in insulin resistance, ER stress and inflammation were analyzed. RESULTS: Chronic and acute palmitate exposure significantly reduced GLUT4 protein (~ 39%, P < 0.01) and its mRNA (18%, P < 0.01) expression. Only acute palmitate treatment increased GRP78 (28%, P < 0.05), PERK (98%, P < 0.01), eIF-2A (35%, P < 0.01), IRE1a (60%, P < 0.05) and TRAF2 (23%, P < 0.05) protein content, and PERK phosphorylation (106%, P < 0.001), but did not elicit eIF-2A, IKK phosphorylation or increased XBP1 nuclear content. Additionally, acute and chronic palmitate increased NFKB p65 nuclear content (~ 30%, P < 0.05) and NFKB binding activity to Slc2a4 gene promoter (~ 45%, P < 0.05). CONCLUSION: Different pathways are activated in acute and chronic palmitate induced-repression of Slc2a4/GLUT4 expression. This regulation involves activation of initial component of ER stress, such as the formation of a IRE1a-TRAF2-IKK complex, and converges to NFKB-induced repression of Slc2a4/GLUT4. These results link ER stress, inflammation and insulin resistance in L6 cells.

Prolonged fasting elicits increased hepatic triglyceride accumulation in rats born to dexamethasone-treated mothers.

We investigated the effect of dexamethasone during the last week of pregnancy on glucose and lipid metabolism in male offspring. Twelve-week old offspring were evaluated after fasting for 12-hours (physiological) and 60-hours (prolonged). Physiological fasting resulted in glucose intolerance, decreased glucose clearance after pyruvate load and increased PEPCK expression in rats born to dexamethasone-treated mothers (DEX). Prolonged fasting resulted in increased glucose tolerance and increased glucose clearance after pyruvate load in DEX. These modulations were accompanied by accumulation of hepatic triglycerides (TG). Sixty-hour fasted DEX also showed increased citrate synthase (CS) activity, ATP citrate lyase (ACLY) content, and pyruvate kinase 2 (pkm2), glucose transporter 1 (slc2a1) and lactate dehydrogenase-a (ldha) expressions. Hepatic AKT2 was increased in 60-hour fasted DEX, in parallel with reduced miRNAs targeting the AKT2 gene. Altogether, we show that metabolic programming by prenatal dexamethasone is characterized by an unexpected hepatic TG accumulation during prolonged fasting. The underlying mechanism may depend on increased hepatic glycolytic flux due to increased pkm2 expression and consequent conversion of pyruvate to non-esterified fatty acid synthesis due to increased CS activity and ACLY levels. Upregulation of AKT2 due to reduced miRNAs may serve as a permanent mechanism leading to increased pkm2 expression.

Metabolic Impact of Light Phase-Restricted Fructose Consumption Is Linked to Changes in Hypothalamic AMPK Phosphorylation and Melatonin Production in Rats.

Recent studies show that the metabolic effects of fructose may vary depending on the phase of its consumption along with the light/dark cycle. Here, we investigated the metabolic outcomes of fructose consumption by rats during either the light (LPF) or the dark (DPF) phases of the light/dark cycle. This experimental approach was combined with other interventions, including restriction of chow availability to the dark phase, melatonin administration or intracerebroventricular inhibition of adenosine monophosphate-activated protein kinase (AMPK) with Compound C. LPF, but not DPF rats, exhibited increased hypothalamic AMPK phosphorylation, glucose intolerance, reduced urinary 6-sulfatoxymelatonin (6-S-Mel) (a metabolite of melatonin) and increased corticosterone levels. LPF, but not DPF rats, also exhibited increased chow ingestion during the light phase. The mentioned changes were blunted by Compound C. LPF rats subjected to dark phase-restricted feeding still exhibited increased hypothalamic AMPK phosphorylation but failed to develop the endocrine and metabolic changes. Moreover, melatonin administration to LPF rats reduced corticosterone and prevented glucose intolerance. Altogether, the present data suggests that consumption of fructose during the light phase results in out-of-phase feeding due to increased hypothalamic AMPK phosphorylation. This shift in spontaneous chow ingestion is responsible for the reduction of 6-S-Mel and glucose intolerance.

Therapy with resveratrol attenuates obesity-associated allergic airway inflammation in mice.

Obesity and insulin resistance have been associated with deterioration in asthma outcomes. High oxidative stress and deficient activation of AMP-activated protein kinase (AMPK) have emerged as important regulators linking insulin resistance and inflammation. This study aimed to evaluate the effects of resveratrol on obesity-associated allergic pulmonary inflammation. Male C57/Bl6 mice fed with high-fat diet to induce obesity (obese group) or standard-chow diet (lean group) were treated or not with resveratrol (100mg/kg/day, two weeks). Mice were sensitized and challenged with ovalbumin (OVA). At 48h thereafter, bronchoalveolar lavage fluid was performed, and lungs collected for morphological studies and Western blot analysis. Treatment of obese mice with resveratrol significantly reduced hyperglycemia and insulin resistance, as well as the body measures (body mass, fat mass, % fat, and body area). OVA-challenge promoted a higher increase in pulmonary eosinophil infiltration in obese compared with lean mice, which was nearly abrogated by resveratrol treatment. Resveratrol markedly increased the phosphorylated AMPK expression in lung tissues of obese compared with lean mice. Resveratrol reduced the p47phox expression and reactive-oxygen species (ROS) production, and elevated the superoxide dismutase (SOD) levels in lung tissues of obese mice. The increased pulmonary levels of TNF-α and inducible nitric oxide synthase (iNOS) in obese mice were also normalized after resveratrol treatment. In lean mice, resveratrol failed to affect the levels of fasting glucose, p47phox, ROS levels, TNF-α, iNOS and phosphorylated AMPK. Resveratrol exhibits protective effects in obesity-associated lung inflammation that is accompanied by local AMPK activation and antioxidant property.

Vascular Damage in Resistant Hypertension: TNF-Alpha Inhibition Effects on Endothelial Cells.

Inflammatory cytokines have been associated with the pathophysiology of hypertension and target organ damage (TOD). Resistant hypertensive patients (RHTN) are characterized by poor blood pressure control and higher prevalence of TOD. This study evaluated the relationship between plasma levels of TNF-α and arterial stiffness (pulse wave velocity-PWV) in 32 RHTN and 19 normotensive subjects. Moreover, we investigated the effect of TNF-α inhibition on human endothelial cells (HUVECs) incubated with serum from RHTN and normotensive subjects. HUVECs containing serum obtained from normotensive (n = 8) and hypertensive (n = 8) individuals were treated with TNF-α inhibitor (infliximab). Cell suspensions were used for measurement of DNA fragmentation and reactive oxygen species (ROS) content. RHTN patients showed higher levels of TNF-α compared to normotensive subjects, as well as higher PWV. Positive correlation was found between TNF-α levels and PWV measures in the whole group. HUVECs incubated with serum from RHTN showed increased cell apoptosis and higher ROS content compared to normotensive subjects. Infliximab attenuated the apoptosis of HUVECs incubated with serum from RHTN, but no effect in ROS production was observed. Our findings suggest that TNF-α might mediate, at least in part, vascular damage in resistant hypertension.

Decreased expression of stem cell markers by simvastatin in 7,12-dimethylbenz(a)anthracene (DMBA)-induced breast cancer.

Simvastatin, a competitive inhibitor of HMG-CoA reductase widely used in the treatment and prevention of hyperlipidemia-related diseases, has recently been associated to in vitro anticancer stem cell (CSC) actions. However, these effects have not been confirmed in vivo. To assess in vivo anti-CSC effects of simvastatin, female Sprague-Dawley rats with 7,12-dimethyl-benz(a)anthracene (DMBA)-induced mammary cancer and control animals were treated for 14 days with either simvastatin (20 or 40 mg/kg/day) or soybean oil (N = 60). Tumors and normal breast tissues were removed for pathologic examination and immunodetection of CSC markers. At 40 mg/kg/day, simvastatin significantly reduced tumor growth and the expression of most CSC markers. The reduction in tumor growth (80%) could not be explained solely by the decrease in CSCs, since the latter accounted for less than 10% of the neoplasia (differentiated cancer cells were also affected). Stem cells in normal, nonneoplastic breast tissues were not affected by simvastatin. Simvastatin was also associated with a significant decrease in proliferative activity but no increase in cell death. In conclusion, this is the first study to confirm simvastatin anti-CSC actions in vivo, further demonstrating that this effect is specific for neoplastic cells, but not restricted to CSCs, and most likely due to inhibition of cell proliferation.