In vivo and in silico study of antioxidant and anti-inflammatory effects on the liver-spleen axis of microencapsulated brewers' spent grain peptides.

Metabolic syndrome (MS) is a cluster of risk factors for the development of cardiovascular disease and type 2 diabetes mellitus. Some dietary bioactive compounds such as peptides can exert dual antioxidant and anti-inflammatory effects. The aim of this study was to analyze the effects of microencapsulated brewers' spent grain peptides (BSG-P-MC) on hepatic injury, lipid peroxidation, oxidative stress and inflammation in the liver-spleen axis in Wistar rats fed with a sucrose-rich diet (SRD). Male rats received for 100 days a reference diet (RD), SRD or RD and SRD containing 700 mg per kg body weight per day of BSG-P-MC. The results demonstrated that BSG-P-MC reversed injury, lipid peroxidation, and oxidative stress in the liver. For the spleen, BSG-P-MC decreased the levels of lipid peroxidation, CAT activity, NFκB, PAI-1 and F4/80 protein mass levels with respect to the SRD-fed rats. Three peptides identified by LC-MS/MS from BSG-P-MC after in vitro gastrointestinal digestion showed high in silico free radical scavenging activity (LPRDPYVDPMAPLPR, ANLPRDPYVDPMAPLPRSGPE and ANLPRDPYVDPMAPLPR). Moreover, two identified peptides presented high in silico anti-inflammatory properties (LTIGDTVPNLELDSTHGKIR and VDPDEKDAQGQLPSRT). This study is the first report of antioxidant and anti-inflammatory properties of microencapsulated BSG-peptides exerted in the liver-spleen axis in a MS rodent model.

Microencapsulated bioactive peptides from brewer's spent grain promotes antihypertensive and antidiabetogenic effects on a hypertensive and insulin-resistant rat model.

The effects of microcapsules containing brewer's spent grain (BSG) peptides were evaluated on a hypertensive/insulin-resistant rat model induced by a sucrose-rich diet (SRD) administration. Animals received for 100 days the control diet (CD), SRD, and CD and SRD diets supplemented with microencapsulated peptides (CD-P and SRD-P). During the experimental period, blood pressure was monitored. Glycemia, tissue glycogen content, nitric oxide, and the activity of enzymes related to hypertensive and diabetogenic mechanisms were determined. The consumption of SRD caused hypertensive and hyperglycemic effects compared to CD. However, the SRD-P group presented lower systolic pressure at the middle of ingestion, achieving similar values than the CD. The SRD-P rats decreased all enzymes' activities compared to the SRD reaching the values of CD, except for those of α-amylase in cecal content and DPP-IV in serum. It was possible to corroborate potential antihypertensive and antidiabetogenic in vivo effects of the microencapsulated BSG peptides. PRACTICAL APPLICATIONS: Brewer's spent grain (BSG) is the main waste obtained from brewing industry. Bioactive peptides obtained after an enzymatic hydrolysis of proteins with in vitro antihypertensive and antidiabetogenic activity have been described. However, to corroborate the action of these bioactive peptides, in vivo studies are necessary. In the present work, microcapsules containing bioactive peptides from BSG were administered on the rat model with induced hypertension and insulin-resistance, corroborating an in vivo antihypertensive and antidiabetogenic effects by inhibition of enzymes related with blood pressure regulation and glucose metabolism. This work demonstrated that microcapsules of BSG peptides could be included into functional foods formulations, or used as dietary supplement for improving health and the prevention of non-communicable diseases, adding value to the brewing process by-product.

Effects of Salvia hispanica L. (chia) seed on blood coagulation, endothelial dysfunction and liver fibrosis in an experimental model of Metabolic Syndrome.

The aim of this study was to analyze blood coagulation, endothelial dysfunction and liver fibrosis in an experimental model of Metabolic Syndrome (MS) induced by chronic administration of a sucrose-rich diet (SRD) and to evaluate the effects of chia seed as a therapeutic strategy. Male Wistar rats were fed with a reference diet (RD) - 6 months - or a SRD - 3 months. Then, the last group was randomly divided into two subgroups. One subgroup continued receiving the SRD for up to 6 months and the other was fed with a SRD where whole chia seed was incorporated as the source of dietary fat for the next 3 months (SRD + CHIA). Results showed that rats fed a SRD for a long period of time develop dyslipidemia, visceral adiposity, insulin resistance, and a hypercoagulable and hypofibrinolytic basal state. Hepatic VCAM-1 (main adhesion molecules involved in endothelial dysfunction) expression was significantly increased. In addition, the SRD group presented hepatic steatosis, a significant increase in interstitial collagen deposition and hydroxyproline content. Liver TGF-ß1 (a key cytokine involved in fibrogenesis) levels increased and a negative correlation with PPARα protein mass levels was found. The administration of chia seed for 3 months reversed dyslipidemia, visceral adiposity and insulin resistance. Platelet count, coagulation parameters and plasma fibrinogen levels were normalized. In the liver tissue, VCAM-1 expression, steatosis, interstitial collagen deposition and the hydroxyproline content decreased. TGF-ß1 expression was decreased and this was associated with an increase in the PPARα protein levels. The present study showed new aspects in the progression from liver steatosis to fibrosis in dyslipidemic insulin-resistant rats chronically fed a sucrose-rich diet. Chia seed supplementation could be used as a functional food and a potential dietary strategy to prevent or ameliorate disorders related to atherothrombotic cardiovascular events and NASH.

Salvia hispanica L. (chia) seed promotes body fat depletion and modulates adipocyte lipid handling in sucrose-rich diet-fed rats.

The aim of this study was to analyze the effects of Salvia hispanica L. (chia) seed upon metabolic pathways that play a key role in adipose tissue lipid handling which could be involved in visceral adiposity reduction developed in rats fed a sucrose-rich diet (SRD). Male Wistar rats were fed with a reference diet (RD) -6 months- or SRD-3 months. Then, the last group was randomly divided into two subgroups. One subgroup continued receiving the SRD up to 6 months and the other was fed with a SRD where whole chia seed was incorporated as the source of dietary fat for the next 3 months (SRD + CHIA). Results showed that chia seed in the SRD-fed rat reduced the abdominal and thoracic circumferences, carcass fat content, adipose tissue weights, and visceral adiposity index. This was accompanied by an improvement in insulin sensitivity and plasma lipid profile. In epididymal adipose tissue, the decreased fat cell triglyceride content was associated with a reduction in both, FAT/CD 36 plasma membrane levels and the fat synthesis enzyme activities. There were not changes in oxidative CPT enzyme activities. PKCß and the precursor and mature forms of SREBP-1 protein levels were decreased, while pAMPK was increased. Our findings suggest that chia seed supplementation can modulate essential pathways of lipid metabolism in adipose tissue, contributing to reduced visceral fat accumulation in SRD-fed rats.

Induction of Lysosome Membrane Permeabilization as a Therapeutic Strategy to Target Pancreatic Cancer Stem Cells.

Despite significant efforts to improve pancreatic ductal adenocarcinoma (PDAC) clinical outcomes, overall survival remains dismal. The poor response to current therapies is partly due to the existence of pancreatic cancer stem cells (PaCSCs), which are efficient drivers of PDAC tumorigenesis, metastasis and relapse. To find new therapeutic agents that could efficiently kill PaCSCs, we screened a chemical library of 680 compounds for candidate small molecules with anti-CSC activity, and identified two compounds of a specific chemical series with potent activity in vitro and in vivo against patient-derived xenograft (PDX) cultures. The anti-CSC mechanism of action of this specific chemical series was found to rely on induction of lysosomal membrane permeabilization (LMP), which is likely associated with the increased lysosomal mass observed in PaCSCs. Using the well characterized LMP-inducer siramesine as a tool molecule, we show elimination of the PaCSC population in mice implanted with tumors from two PDX models. Collectively, our approach identified lysosomal disruption as a promising anti-CSC therapeutic strategy for PDAC.

Lipotoxicidad en músculo esquelético y su relación con la resistencia insulínica. Estudios en un modelo experimental de Síndrome metabólico / Skeletal muscle lipotoxicity and its relationship with insulin resistance. Studies in an experimental model of metabolic syndrome

Introducción: el acúmulo de lípidos en el músculo esquelético se encuentra estrechamente vinculado con el desarrollo de la resistencia insulínica. Esta última cumple un rol patogénico central en el desarrollo de numerosos desórdenes metabólicos incluidos en el síndrome metabólico. Objetivos: analizar algunas vías metabólicas implicadas en el acúmulo de lípidos en el músculo esquelético y su asociación con la resistencia insulínica en un modelo experimental que mimetiza el fenotipo del síndrome metabólico humano. Materiales y métodos: ratas macho Wistar recibieron una dieta control (DC) o una dieta rica en sacarosa (DRS) durante seis meses. Al final del período experimental se analizó en músculo esquelético gastrocnemio: contenido de triglicéridos (TG), acil-CoA de cadena larga y diacilglicerol, actividad enzimática carnitina palmitoil transferasa muscular (M-CPT1, M-CPT2 y M-CPT total) y masa proteica del PPARα, AMPK y AMPKp. Se determinaron los niveles séricos de TG, AGNE, glucosa, insulina, TNFα y adiponectina. La sensibilidad insulínica se midió por la técnica clamp euglucémica-hiperinsulinémica. Resultados: en los animales alimentados con DRS la dislipemia, hiperglucemia moderada, insensibilidad insulínica e incremento del contenido de especies lipídicas en el músculo esquelético se acompañaron de una disminución en la actividad enzimática M-CPT1 y M-CPT total, y un descenso de la masa proteica del PPARα. Además se observó una reducción de la masa proteica de la AMPKp, la cual se correlacionó con bajos niveles de adiponectina y elevados niveles de TNFα séricos. Conclusiones: los resultados aportan nuevos datos sobre algunos mecanismos involucrados en el desarrollo de la lipotoxicidad en el músculo esquelético en ratas dislipémicas insulinorresistentes

Introduction: the skeletal muscle lipid accumulation is closely linked to the development of insulin resistance. The latter plays a central pathogenic role in the development of numerous metabolic disorders included in the metabolic syndrome. Objectives: to analyze some metabolic pathways involved in the skeletal muscle lipid accumulation and its association with insulin resistance in an experimental model that mimics the phenotype of the human metabolic syndrome. Materials and methods: male Wistar rats received a control diet (CD) or a sucrose rich diet (SRD) for six months. At the end of the experimental period, in gastrocnemius skeletal muscle were analyzed: triglyceride (TG), long chain acyl-CoA and diacylglycerol (DAG) contents, muscle carnitine palmitoyl transferase enzymes activities (M-CPT1, M-CPT2 and total M-CPT) and protein mass levels of PPARα, AMPK and AMPKp. Serum levels of TG, AGNE, glucose and insulin, TNFα and adiponectin were determined. Insulin sensitivity was measured by the euglycemic-hyperinsulinemic clamp technique. Results: in SRD fed animals, dyslipidemia, moderate hyperglycemia, insulin insensitivity and the increased content of lipid species in the skeletal muscle were accompanied by a decrease in the enzymes activities of both M-CPT1 and total M-CPT and protein mass levels of PPARα. In addition, a reduction in the protein mass levels of AMPKp was observed, which was correlated with low serum levels of adiponectin and high levels of TNFα. Conclusions: the results provide new data on some mechanisms involved in the development of lipotoxicity in skeletal muscle in insulin resistant dyslipidemic rats

Dietary fish oil ameliorates adipose tissue dysfunction in insulin-resistant rats fed a sucrose-rich diet improving oxidative stress, peroxisome proliferator-activated receptor γ and uncoupling protein 2.

This work aims to assess the possible beneficial effects of dietary fish oil (FO) on the pre-existing adipose tissue dysfunction through the improvement or reversion of the mechanisms underlying oxidative stress and pro-inflammatory cytokines in dyslipemic insulin-resistant rats. Wistar rats were fed a sucrose rich diet (SRD) for 6 months. After that half of the animals continued with the SRD until month 8 while in the other half corn oil was replaced by FO for 2 months (SRD + FO). A reference group consumed a control diet all the time. In an epididymal fat pad, we analyzed antioxidant and oxidant enzyme activities, ROS content, glutathione redox state, the protein level of peroxisome proliferator-activated receptor gamma (PPARγ) and the expression and protein levels of uncoupling protein 2 (UCP2) as well as oxidative stress biomarkers and TNF-α and IL-6 plasma levels. Besides these, insulin sensitivity and the composition of fatty acid phospholipids of adipose tissue were measured. Compared with the SRD the SRD + FO fed group showed a decrease of fat pad weight and the antioxidant and oxidant enzyme activities and ROS content returned to control values along with normal plasma TNF-α and IL-6 levels. FO normalized both the decrease of PPARγ protein and the increase of protein and expression of UCP2. Furthermore, FO increased the n-3/n-6 fatty acid ratio in the adipose tissue phospholipids and normalized dyslipidemia and insulin resistance. Finally, these findings reinforce the view that dietary FO may exert a beneficial effect in ameliorating the dyslipidemia and insulin resistance in this animal model.

Discovery of (7R)-14-cyclohexyl-7-{[2-(dimethylamino)ethyl](methyl) amino}-7,8-dihydro-6H-indolo[1,2-e][1,5]benzoxazocine-11-carboxylic acid (MK-3281), a potent and orally bioavailable finger-loop inhibitor of the hepatitis C virus NS5B polymerase.

Infections caused by hepatitis C virus (HCV) are a significant world health problem for which novel therapies are in urgent demand. The polymerase of HCV is responsible for the replication of viral genome and has been a prime target for drug discovery efforts. Here, we report on the further development of tetracyclic indole inhibitors, binding to an allosteric site on the thumb domain. Structure-activity relationship (SAR) studies around an indolo-benzoxazocine scaffold led to the identification of compound 33 (MK-3281), an inhibitor with good potency in the HCV subgenomic replication assay and attractive molecular properties suitable for a clinical candidate. The compound caused a consistent decrease in viremia in vivo using the chimeric mouse model of HCV infection.

ß-Cell adaptation/dysfunction in an animal model of dyslipidemia and insulin resistance induced by the chronic administration of a sucrose-rich diet.

Glucose stimulated insulin secretion (GSIS) was different in rats chronically fed a sucrose-rich diet (SRD) for 3 or 30 wk. This work proposes possible mechanisms underlying insulin secretion changes from ß-cell throughout these feeding periods. In isolated islets of rats fed the SRD or a control diet (CD) we examined: 1- the glucokinase and hexokinase activities and their protein mass expression; 2- pyruvate dehydrogenase activity; 3- uncoupling protein 2 (UCP2) and peroxisome proliferators-activated receptor γ (PPAR γ) protein mass expression. At 3 wk on diet the SRD-fed rats showed: a marked increase in the first peak of GSIS; increased glucokinase protein mass expression without changes in glucokinase and hexokinase activities; increased PPARγ protein mass expression without changes in the UCP2 protein mass expression. No changes in either glucose oxidation and triglyceride content within the ß-cell were observed. After 30 wk of feeding, a significant decrease of both glucokinase activity and its protein mass expression was accompanied by altered glucose oxidation, a triglyceride increase within the ß-cell and a significant increase of PPARγ and UCP2 protein mass expression. Moreover GSIS depicted an absence of the first peak with an increase in the second phase. Finally, the SRD chronic administration altered GSIS by different mechanisms depending on the time on diet. At an early stage, the increased protein mass expression of the glucokinase and a fatty acid cooperative effect inducing PPARγ expression seem to be the mechanisms involved. At a late stage, glucolipotoxicity appears to be the cellular mechanism contributing to progressive ß-cell dysfunction.

Discovery of pentacyclic compounds as potent inhibitors of hepatitis C virus NS5B RNA polymerase.

We report a new series of inhibitors for hepatitis C virus NS5B RNA polymerase containing a constrained pentacyclic scaffold. Our SAR studies led to the identification of hexahydroindolo[2,1-a]pyrrolo[3,2-d][2]benzazepines exposing basic groups. The compounds displayed a high activity in the enzyme assay and displayed good activity in the cell-based (replicon) assay in the presence of serum proteins.

Discovery and synthesis of HIV integrase inhibitors: development of potent and orally bioavailable N-methyl pyrimidones.

The human immunodeficiency virus type-1 (HIV-1) encodes three enzymes essential for viral replication: a reverse transcriptase, a protease, and an integrase. The latter is responsible for the integration of the viral genome into the human genome and, therefore, represents an attractive target for chemotherapeutic intervention against AIDS. A drug based on this mechanism has not yet been approved. Benzyl-dihydroxypyrimidine-carboxamides were discovered in our laboratories as a novel and metabolically stable class of agents that exhibits potent inhibition of the HIV integrase strand transfer step. Further efforts led to very potent compounds based on the structurally related N-Me pyrimidone scaffold. One of the more interesting compounds in this series is the 2-N-Me-morpholino derivative 27a, which shows a CIC95 of 65 nM in the cell in the presence of serum. The compound has favorable pharmacokinetic properties in three preclinical species and shows no liabilities in several counterscreening assays.

An enantioselective total synthesis of (+)- and (-)-saudin. Determination of the absolute configuration.

A short efficient enantioselective synthesis of both (+)- and (-)-saudin, a naturally occurring hypoglycemic diterpene, is described. This synthesis establishes the absolute configuration of natural (-)-saudin for the first time. The key steps include the enantioselective construction of a dimethyl Hagemann's ester by an asymmetric Michael reaction and establishment of the key 1,3 disposed quaternary centers by means of a novel Ti(IV) promoted Claisen rearrangement. The assembly of the polycyclic ketal skeleton was likely under kinetic control proceeding via formation of the C1oxygen-C7 bond through an oxonium ion intermediate in the final stage.