Pecans and Its Polyphenols Prevent Obesity, Hepatic Steatosis and Diabetes by Reducing Dysbiosis, Inflammation, and Increasing Energy Expenditure in Mice Fed a High-Fat Diet.

Pecans (Carya illinoinensis) are considered a functional food due to the high content of polyunsaturated fatty acids, dietary fiber and polyphenols. To determine the effect of whole pecans (WP) or a pecan polyphenol (PP) extract on the development of metabolic abnormalities in mice fed a high-fat (HF) diet, we fed C57BL/6 mice with a Control diet (7% fat), HF diet (23% fat), HF containing 30% WP or an HF diet supplemented with 3.6 or 6 mg/g of PP for 18 weeks. Supplementation of an HF diet with WP or PP reduced fat mass, serum cholesterol, insulin and HOMA-IR by 44, 40, 74 and 91%, respectively, compared to the HF diet. They also enhanced glucose tolerance by 37%, prevented pancreatic islet hypertrophy, and increased oxygen consumption by 27% compared to the HF diet. These beneficial effects were associated with increased thermogenic activity in brown adipose tissue, mitochondrial activity and AMPK activation in skeletal muscle, reduced hypertrophy and macrophage infiltration of subcutaneous and visceral adipocytes, reduced hepatic lipid content and enhanced metabolic signaling. Moreover, the microbial diversity of mice fed WP or PP was higher than those fed HF, and associated with lower circulating lipopolysaccharides (~83-95%). Additionally, a 4-week intervention study with the HF 6PP diet reduced the metabolic abnormalities of obese mice. The present study demonstrates that WP or a PP extract prevented obesity, liver steatosis and diabetes by reducing dysbiosis, inflammation, and increasing mitochondrial content and energy expenditure. Pecan polyphenols were mainly condensed tannin and ellagic acid derivatives including ellagitannins as determined by LC-MS. Herein we also propose a model for the progression of the HF diet-mediated metabolic disorder based on early and late events, and the possible molecular targets of WP and PP extract in preventive and intervention strategies. The body surface area normalization equation gave a conversion equivalent to a daily human intake dose of 2101-3502 mg phenolics that can be obtained from 110-183 g pecan kernels/day (22-38 whole pecans) or 21.6-36 g defatted pecan flour/day for an average person of 60 kg. This work lays the groundwork for future clinical studies.

Ethical considerations in animal research: the principle of 3r's

In the last century, progress in the knowledge of human diseases, their diagnosis and treatment have grown exponentially, due in large part to the introduction and use of laboratory animals. Along with this important progress, the need to provide training and guidance to the scientific community in all aspects related to the proper use of experimental animals has been indispensable. Animal research committees play a primary role in evaluating experimental research protocols, from their feasibility to the rational use of animals, but above all in seeking animal welfare. The Institutional Committee for the Care and Use of Animals (IACUC) has endeavored to share several relevant aspects in conducting research with laboratory animals. Here, we present and discuss the topics that we consider of utmost importance to take in the account during the design of any experimental research protocol, so we invite researchers, technicians, and undergraduate and graduate students to dive into the fascinating subject of proper animal care and use for experimentation. The main intention of these contributions is to sensitize users of laboratory animals for the proper and rational use of them in experimental research, as well as to disseminate the permitted and unpermitted procedures in laboratory animals. In the first part, the significance of experimental research, the main functions of IACUC, and the principle of the three R's (replacement, reduction, and refinement) are addressed.

PPARα/RXRα downregulates amino acid catabolism in the liver via interaction with HNF4α promoting its proteasomal degradation.

The preservation of body proteins is essential to guarantee their functions in organisms. Therefore, the utilization of amino acids as energy substrates is regulated by a precise fine-tuned mechanism. Recent evidence suggests that the transcription factors peroxisome proliferator-activated receptor alpha (PPARα) and hepatocyte nuclear factor 4 alpha (HNF4α) are involved in this regulatory mechanism. Thus, the aim of this study was to determine how these transcription factors interact to regulate the expression of amino acid catabolism genes. In vivo studies using PPARα-knockout mice (Pparα-null) fed different amounts of dietary protein showed that in the absence of PPARα, there was a significant increase in HNF4α abundance in the liver, which corresponded with an increase in amino acid catabolizing enzyme (AACE) expression and the generation of increased amounts of postprandial urea. Moreover, this effect was proportional to the increase in dietary protein consumed. Chromatin immunoprecipitation assays showed that HNF4α can bind to the promoter of AACE serine dehydratase (SDS), an effect that was potentiated by dietary protein in the Pparα-null mice. The mechanistic studies revealed that the presence of retinoid X receptor alpha (RXRα) is essential to repress HNF4α activity in the presence of PPARα, and this interaction accelerates HNF4α degradation via the proteasome pathway. These results showed that PPARα can downregulate liver amino acid catabolism in the presence of RXRα by inhibiting HNF4α activity.

Goat's Milk Intake Prevents Obesity, Hepatic Steatosis and Insulin Resistance in Mice Fed A High-Fat Diet by Reducing Inflammatory Markers and Increasing Energy Expenditure and Mitochondrial Content in Skeletal Muscle.

Goat's milk is a rich source of bioactive compounds (peptides, conjugated linoleic acid, short chain fatty acids, monounsaturated and polyunsaturated fatty acids, polyphenols such as phytoestrogens and minerals among others) that exert important health benefits. However, goat's milk composition depends on the type of food provided to the animal and thus, the abundance of bioactive compounds in milk depends on the dietary sources of the goat feed. The metabolic impact of goat milk rich in bioactive compounds during metabolic challenges such as a high-fat (HF) diet has not been explored. Thus, we evaluated the effect of milk from goats fed a conventional diet, a conventional diet supplemented with 30% Acacia farnesiana (AF) pods or grazing on metabolic alterations in mice fed a HF diet. Interestingly, the incorporation of goat's milk in the diet decreased body weight and body fat mass, improved glucose tolerance, prevented adipose tissue hypertrophy and hepatic steatosis in mice fed a HF diet. These effects were associated with an increase in energy expenditure, augmented oxidative fibers in skeletal muscle, and reduced inflammatory markers. Consequently, goat's milk can be considered a non-pharmacologic strategy to improve the metabolic alterations induced by a HF diet. Using the body surface area normalization method gave a conversion equivalent daily human intake dose of 1.4 to 2.8 glasses (250 mL per glass/day) of fresh goat milk for an adult of 60 kg, which can be used as reference for future clinical studies.

Genistein increases the thermogenic program of subcutaneous WAT and increases energy expenditure in mice.

White adipose tissue (WAT) can differentiate into beige adipose tissue by the browning process. Some polyphenols, including isoflavones, particularly genistein, are suggested to increase the expression of browning markers. There is evidence that consumption of genistein can attenuate body weight gain and improve glucose tolerance and blood lipid levels. The aim of the present study was to investigate the potential mechanisms of stimulation by which genistein activates the browning of WAT. We studied the stimulation of the expression of browning markers in the following models: mice fed genistein; preadipocytes from 3 T3-L1 cells; and the stromal vascular fraction (SVF) from the inguinal adipose tissue of mice. The results indicated that genistein can stimulate the browning process by at least two mechanisms. An indirect mechanism was involved in the induction of PGC-1α/FNDC5 in skeletal muscle leading to an increase in the myokine irisin. In preadipocytes, irisin was able to increase the expression of Ucp1 and Tmem26, markers of browning, to increase energy expenditure. Interestingly, genistein was also able to activate browning by a direct mechanism. Incubation of preadipocytes with genistein increased UCP1 expression as well as some biomarkers of browning in a concentration-dependent manner, possibly via phosphorylation of AMPK. The effect of genistein was accompanied by an increase in the number of mitochondria as well as in the maximum respiration rate of the adipocytes. In conclusion, this study indicated that genistein can increase energy expenditure by stimulating the browning process directly in preadipocytes and indirectly by increasing the circulating levels of irisin.

PPARα Downregulates Hepatic Glutaminase Expression in Mice Fed Diets with Different Protein:Carbohydrate Ratios.

BACKGROUND: Glutamine is catabolized in the liver by glutaminase 2 (GLS2). Evidence suggests that peroxisome proliferator-activated receptor α (PPARα) represses the expression of several amino acid-catabolizing enzymes, but for Gls2 this is unknown. OBJECTIVE: The aim of the study was to assess whether PPARα regulates Gls2 expression. METHODS: For 8 d, 7-9-wk-old male C57BL/6 wild-type (WT) and Ppara-null mice weighing 23.4 ± 0.5 g were fed diets with different dietary protein:carbohydrate (DP:DCH) ratios (6%:77%, 20%:63%, or 50%:33%). Liver samples were obtained after 16 h of feed deprivation or 3 h of refeeding, and microarrays were performed. Hepatic glutaminase expression was measured by quantitative polymerase chain reaction and Western blotting. Cotransfection analyses in hepatocellular carcinoma cell line (HepG2) cells with PPARα and hepatocyte nuclear factor 4α (HNF4α) expression vectors were performed. RESULTS: The microarray results showed that Gls2 was the only upregulated gene in WT mice, but not in the Ppara-null mice. In the feed-deprived WT mice, the Gls2 mRNA and protein abundances in the 50%:33% group were 2.5- and 1.1-fold greater (P < 0.05), respectively, than those in the 20%:63% group, which were 2.3- and 0.4-fold greater than those in the 6%:77% group (P < 0.01). Gls2 mRNA expression in the 6%:77% group of feed-deprived Ppara-null mice was 33-fold greater than that in the same group of WT mice (P < 0.0001). GLS2 protein abundance in HepG2 cells was 78% greater than that in the controls (P < 0.0001) after HNF4α overexpression, and it was 99% greater after transfection with a short hairpin targeting PPARα. CONCLUSIONS: In Ppara-null mice, Gls2 mRNA expression was greater than in WT mice, regardless of the DP:DCH ratio. In HepG2 cells overexpressing HNF4α, Gls2 expression increased, an effect repressed by overexpression of PPARα. This suggests that Gls2 depends on the PPARα/HNF4α counterregulatory transcriptional control.

Hepatic amino acid-degrading enzyme expression is downregulated by natural and synthetic ligands of PPARα in rats.

Body nitrogen retention is dependent on the amount of dietary protein consumed, as well as the fat and carbohydrate content in the diet, due to the modulation of amino acid oxidation. PPARα is a transcription factor involved in the upregulation of the expression of enzymes of fatty acid oxidation. However, the role of putative PPARα response elements (PPREs) in the promoter of several amino acid-degrading enzymes (AADEs) is not known. The aim of this work was to study the effect of the synthetic ligand Wy 14643 and the natural ligands palmitate, oleate, and linoleate in rats fed graded concentrations of dietary protein (6, 20, or 50 g/100 g of total diet) on the expression of the AADEs histidase, serine dehydratase, and tyrosine aminotransferase. Thus, we fed male Wistar rats diets containing 6, 20, or 50% casein for 10 d. The results showed that addition of Wy 14643 to the diet significantly reduced the expression of the AADEs. Furthermore, the incubation of hepatocytes with natural ligands of PPARα or feeding rats with diets containing soybean oil, safflower oil, lard, or coconut oil as sources of dietary fat significantly repressed the expression of the AADEs. Gene reporter assays and mobility shift assays demonstrated that the PPRE located at -482 bp of the histidase gene actively bound PPARα in rat hepatocytes. These data indicate that PPARα ligands may reduce amino acid catabolism in rats.

Promoter characterization and role of CRE in the basal transcription of the rat SNAT2 gene.

Small neutral amino acid transporter 2 (SNAT2) is the most abundant and ubiquitous transporter for zwitterionic short-chain amino acids. The activity of this amino acid transporter is stimulated in vivo or in vitro by glucagon or cAMP analogs. However, it is not known whether the increase in activity at the protein level is due to an increase in SNAT2 gene transcription. Thus, the aim of the present work was to study whether cAMP was able to stimulate SNAT2 gene expression and to localize and characterize the presence of cAMP response elements (CRE) in the promoter that controls the expression of the rat SNAT2 gene. We found that consumption of a high-protein diet that increased serum glucagon concentration or the administration of glucagon or incubation of hepatocytes with forskolin increased the SNAT2 mRNA level. We then isolated the 5' regulatory region of the SNAT2 gene and determined that the transcriptional start site was located 970 bp upstream of the translation start codon. We identified two potential CRE sites located at -354 and -48 bp. Our results, using deletion analysis of the 5' regulatory region of the SNAT2 gene, revealed that the CRE site located at -48 bp was fully responsible for SNAT2 regulation by cAMP. This evidence was strongly supported by mutation of the CRE site and EMSA and ChIP analysis. Alignment of rat, mouse, and human sequences revealed that this CRE site is highly conserved among species, indicating its essential role in the regulation of SNAT2 gene expression.

Activation of SIRT1 by resveratrol represses transcription of the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) by deacetylating hepatic nuclear factor 4alpha.

The SIRT1 activators isonicotinamide (IsoNAM), resveratrol, fisetin, and butein repressed transcription of the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (PEPCK-C). An evolutionarily conserved binding site for hepatic nuclear factor (HNF) 4alpha (-272/-252) was identified, which was required for transcriptional repression of the PEPCK-C gene promoter caused by these compounds. This site contains an overlapping AP-1 binding site and is adjacent to the C/EBP binding element (-248/-234); the latter is necessary for hepatic transcription of PEPCK-C. AP-1 competed with HNF4alpha for binding to this site and also decreased HNF4alpha stimulation of transcription from the PEPCK-C gene promoter. Chromatin immunoprecipitation experiments demonstrated that HNF4alpha and AP-1, but not C/EBPbeta, reciprocally bound to this site prior to and after treating HepG2 cells with IsoNAM. IsoNAM treatment resulted in deacetylation of HNF4alpha, which decreased its binding affinity to the PEPCK-C gene promoter. In HNF4alpha-null Chinese hamster ovary cells, IsoNAM and resveratrol failed to repress transcription from the PEPCK-C gene promoter; overexpression of HNF4alpha in Chinese hamster ovary cells re-established transcriptional inhibition. Exogenous SIRT1 expression repressed transcription, whereas knockdown of SIRT1 by RNA interference reversed this effect. IsoNAM decreased the level of mRNA for PEPCK-C but had no effect on mRNA for glucose-6-phosphatase in AML12 mouse hepatocytes. We conclude that SIRT1 activation inhibited transcription of the gene for PEPCK-C in part by deacetylation of HNF4alpha. However, SIRT1 deacetylation of other key regulatory proteins that control PEPCK-C gene transcription also likely contributed to the inhibitory effect.

Characterization and regulation of the gene expression of amino acid transport system A (SNAT2) in rat mammary gland.

Amino acid transport via system A plays an important role during lactation, promoting the uptake of small neutral amino acids, mainly alanine and glutamine. However, the regulation of gene expression of system A [sodium-coupled neutral amino acid transporter (SNAT)2] in mammary gland has not been studied. The aim of the present work was to understand the possible mechanisms of regulation of SNAT2 in the rat mammary gland. Incubation of gland explants in amino acid-free medium induced the expression of SNAT2, and this response was repressed by the presence of small neutral amino acids or by actinomycin D but not by large neutral or cationic amino acids. The half-life of SNAT2 mRNA was 67 min, indicating a rapid turnover. In addition, SNAT2 expression in the mammary gland was induced by forskolin and PMA, inducers of PKA and PKC signaling pathways, respectively. Inhibitors of PKA and PKC pathways partially prevented the upregulation of SNAT2 mRNA during adaptive regulation. Interestingly, SNAT2 mRNA was induced during pregnancy and to a lesser extent at peak lactation. beta-Estradiol stimulated the expression of SNAT2 in mammary gland explants; this stimulation was prevented by the estrogen receptor inhibitor ICI-182780. Our findings clearly demonstrated that the SNAT2 gene is regulated by multiple pathways, indicating that the expression of this amino acid transport system is tightly controlled due to its importance for the mammary gland during pregnancy and lactation to prepare the gland for the transport of amino acids during lactation.

Regulation by glucagon of the rat histidase gene promoter in cultured rat hepatocytes and human hepatoblastoma cells.

Histidase (Hal), the amino acid-degrading enzyme of histidine, is regulated by the protein content of the diet and by hormones such as glucocorticoids and glucagon. However, glucagon can activate the following two possible transduction pathways: protein kinase A (PKA) and protein kinase C (PKC). The aim of this study was to isolate the 5'-flanking region of rat Hal gene to locate possible cAMP- and glucocorticoid-responsive elements and to identify whether the activation of the Hal promoter by glucagon occurs via PKA or PKC. The results showed that glucagon was able to induce Hal expression 1.5-fold in primary hepatocytes. The addition of phorbol 12-myristate,13-acetate (PMA) and forskolin to hepatocytes increased Hal mRNA concentration by 100 and 40%, respectively. To identify the Hal gene regulatory region, a 1248-bp fragment of the 5'-region was obtained. The transcription initiation site was located at 404 bp from ATG. The sequence did not show consensus TATA-like or CAAT-like boxes in the first 100 bp upstream from the transcription start site. The promoter contained six GC rich boxes, seven putative AP1 binding sites, and four glucocorticoid-responsive elements. The putative Hal promoter region was cloned into the pGL3basic vector and transfected into HepG2 cells. Luciferase expression was significantly stimulated by glucagon (0.9-fold), forskolin (0.9-fold), PMA (2.0-fold), and dexamethasone (2.9-fold). This evidence supports that the Hal gene is turned on by glucocorticoids and by glucagon either via PKC or PKA, but prefers the PKA pathway.

[Peroxisome proliferator-activated receptors (PPARs) in obesity and insulin resistance development]. / Los receptores activados por proliferadores de peroxisomas (PPARs) en el desarrollo de obesidad y resistencia a la insulina.

The peroxisome proliferator-activated receptors (PPARs) are a family of nuclear transcription factors that belong to the steroid receptor superfamily. PPARs family includes PPARalpha, PPARbeta/delta, PPARgamma1 and PPARgamma2. PPARs form an heterodimer with the 9-cis retinoic acid receptor (RXR) and bind to response elements present in target genes activated by these transcription factors. PPARs control the expression of genes involved in fatty acid synthesis, oxidation and storage. PPARs are present in most tissues, where PPARalpha is most abundant in liver and skeletal muscle, whereas PPARgamma is found mainly in adipose tissue. Natural ligands for PPARs are polyunsaturated fatty acids (PUFAs) and some eicosanoids, however they are also activated by compounds such as fibrates and thiazolidinediones (TZDs). In this review is shown the different PPARs isoforms, identification, and regulation of their expression and activity. Also shows which are the natural ligands, and the chemical compounds that activate PPARs. Finally, it shows the target genes activated by the different isoforms of PPARs, the metabolic integration between the different PPAR isoforms to maintain a balance between fatty acid synthesis and oxidation and the association with the development of obesity and insulin resistance. Also shows information about the nutritional requirements of PUFAs that are the main natural ligands of PPARs.

Metabolismo de la homocisteína y riesgo de enfermedades cardiovasculares: Importancia del estado nutricio en ácido fólico, vitaminas B6 y B12 / Metabolism of homocysteine and risk of cardiovascular diseass: Importance of folic acid, vitamins B6 and B12 nutritional status

La homocisteína es un aminoácido azufrado que se sintetiza en el organismo a partir de la metionina. Este compuesto puede seguir dos rutas es su metabolismo, la de remetilación y la de transulfuración. La primera da lugar a la regeneración de metionina y la segunda a la formación de cisteína. Debido a su rápida utilización metabólica, este aminoácido se encuentra en bajas concentraciones en el plasma. La homocisteína circula en la sangre como tiol puro en bajas concentraciones y la mayoría se encuentra como disulfuro libre unido a residuos de cisteína de las proteínas. Sin embargo, en el caso de defectos genéticos en alguna de las enzimas del metabolismo de la homocistéina o de deficiencia nutricia de ácido fólico, vitamina B6 y B12, cuyas formas coenzimáticas se requieren para la utilización de homocisteína, se produce una elevación significativa de la concentración de este aminoácido en plasma asociado a un incremento en el desarrollo de enfermedades cardiovasculares. En base a estudios clínicos y epidemiológicos en la actualidad se considera la concentración de homocisteína en plasma como un factor independiente de riesgo de desarrollo de enfermedades aterotrombóticas y cardiovasculares. En la presente revisión se describe el metabolismo de la homocisteína, y la evidencia epidemiológica que muestra la asociación de la homocisteína con la incidencia de enfermedades cardiovasculares, así como valores de referencia. Se indican los mecanísmos a través de los cuales este aminoácido azufrado produce daño vascular. Finalmente, se dan algunas recomendaciones para el tratamiento nutricio de pacientes con hiperhomocisteinemia.

Alanine aminotransferase activity in mammary tissue, muscle and liver of dam rat during lactation and weaning

Transamination reaction is the first step in the catabolism of most of the L-amino acids. Alanine is an important molecule in the inter-organ nitrogen transport, conveying them from muscle to the liver Amino groups from this amino acid are generally first transferred to alfa-ketoglutarate in the cytosol of liver cells to form glutamate and leaving behing the corresponding alfa-keto acid analog. Measurements of the alanine aminotransferase (EC2.6.1.2.) activity were compared in liver, mammary gland an skeletal muscle in virgin, lactating and weaning dam rats. In this study liver was the principal tissue involved in alanine transamination, while muscle showed a reduction in the enzyme activity during lactation. Results indicate an increase in alanine aminotransferase activity in the mammary gland during lactation and weaning when compared with virgin rats. This suggest that mammary gland during lactation is an importan extra-hepatic tissue involved in the metabolism of alanine and probably shunted into hte pathways for amino group metabolism in terms of nitrogen economy