The role of adiponectin and the adipocyte in energy metabolism and inflammation

A series of experiments were conducted to characterize the autocrine role of adiponectin in modulating fatty acid metabolism and inflammation in the pig. In the first study, we cloned and sequenced the porcine adiponectin open reading frame and evaluated the regulation of adiponectin, in vitro and in vivo. The porcine sequence shares approximately 88, 86, 85 and 83% homology with the dog, human, cow and mouse adiponectin, respectively, and 79–83% similarity with dog, human, cow and mouse proteins at the amino acid level, based on the translated porcine sequence and GenBank submissions for the other species. Analysis of serum from very lean vs. a substantially fatter line of pigs indicated that relative circulating adiponectin concentrations are higher (P \u3c 0.01) in lean pigs than in the fatter line, and that the difference is established relatively early in the growth curve. Incubating pig adipocytes for 6 hours with recombinant pig adiponectin also resulted in an approximate 30% reduction (P \u3c 0.05) in lipogenesis compared with adipocytes under basal conditions and with those incubated in the presence of insulin.^ Based on in vivo and in vitro data showing adiponectin is positively correlated with leanness and reduces lipogenesis in porcine adipocytes, experiment investigated adipocyte lipid metabolism. In a series of cell culture studies for experiment two, we further investigated the role of adiponectin in modulating lipid metabolism by measuring mRNA abundance of genes associated with fatty acid synthesis and oxidation in porcine adipocytes. Adiponectin transiently increased (2–6.5 fold, P \u3c 0.05) the expression of acetyl Co-A carboxylase (ACC), AMP activated kinase (AMPK), acyl Co-A oxidase (ACO) and peroxisome proliferator activated receptor-&agr; (PPAR&agr;). After the initial increase in mRNA abundance, there was either a return to initial levels of expression (PPAR&agr;), a significant reduction (AMPK), or a cyclic pattern of expression (ACC, ACO). The abundance of the uncoupling protein-3 (UCP3) transcript was not influenced by adiponectin until the final measure at 360 minutes, at which time it was increased 3.8-fold (P \u3c 0.05). ^ Experiment three was designed to examine anti-inflammatory properties of adiponectin contributing to its insulin sensitizing effects through regulation of the adiponectin receptors, adipoR1 and adipoR2. Cells were incubated ± TNF&agr; or IL-6 (30 ng/mL), with or without pretreatment of 10 &mgr;M AG490, in hyper-glycemic or normo-glycemic conditions for 6 h. There was no effect of cytokines at 5.5 mM glucose for R1 expression. However, there was a trend for a down-regulation of R2 by AG490-TNF&agr; (P \u3c 0.07). At 25 mM glucose, there was an increase in R1 by AG490-TNF&agr; treatment (P = 0.06). R2 was marginally reduced by IL-6, but there was a reduction (P \u3c .01) with AG490 plus IL-6. High glucose caused a reduction of both receptors, whereas TNF&agr; increased both in the high glucose media. R1 (P \u3c 0.1) and R2 (P \u3c 0.05) were further elevated by AG490 plus TNF&agr; in high glucose. Interestingly, IL-6 and AG490 plus IL-6 (P \u3c 0.05) reduced R1 and R2 expression, but only in the high glucose media. Collectively, these data indicate that the effects of TNF&agr; and IL-6 on adiponectin receptor expression are influenced by glucose concentration, and that the JAK-STAT pathway may be a determinant of adiponectin receptor expression.^ In the final experiment, the effects of hyperglycemia on insulin sensitivity in pig adipocytes, and 3T3-L1 adipocytes were examined. In pig adipocytes, concentrations of 25 and 40 mM glucose inhibited insulin stimulated glucose uptake (P \u3c 0.05), that could not be reversed by addition of adiponectin to the culture media. To examine if hyperglycemic conditions were associated with impaired insulin signaling pig adipocytes were cultured in euglycemic and hyperglycemic conditions and insulin receptor and Akt phosphorylation were measured by semiquantitative Western blots. However, insulin resistance in primary pig adipocytes could not be attributed to changes in protein phosphosphorlation of the insulin receptor or its down-stream target Akt (P \u3e 0.05). To examine glycemic regulation of insulin-induced glucose uptake for longer duration the 3T3-L1 mouse derived adipocytes were used. Similar to the effects seen in acute porcine adipocyte cultures hyperglycemia inhibited insulin stimulated 2-deoxyglucose uptake in 3T3-L1 adipocytes (P \u3c 0.05), and 24 hour treatment with adiponectin could not reverse the insulin resistance. Again, difference in phosphorylation of the insulin receptor and Akt could not be detected in hyperglycemic culture cells compared with control cells. Interestingly, although adiponectin did not appear to impact insulin signaling pathways in under hyperglycemic conditions it did have an effect on inflammatory pathways in the adipocyte. Adiponectin reduced the production of intracellular ROS in 3T3-L1 adipocytes cultured in hyperglycemic conditions (P \u3c 0.05). (Abstract shortened by UMI.

Monitoring Muscle Stem Cell Cultures with Impedance Spectroscopy

The aim of this work is to present a new circuit for the real-time monitoring the processes of cellular growth and differentiation of skeletal myoblast cell cultures. An impedance spectroscopy Oscillation-Based technique is proposed for the test circuit, converting the biological system into a voltage oscillator, and avoiding the use of very high performance circuitry or equipment. This technique proved to be successful in the monitoring of cell cultures growth levels and could be useful for determining the degree of differentiation achieved, of practical implications in tissue engineering.Ministerio de Economía y Competitividad TEC2013-46242-C3-1-

Electrical Modeling of the Growth and Differentiation of Skeletal Myoblasts Cell Cultures for Tissue Engineering

In tissue engineering, of utmost importance is the control of tissue formation, in order to form tissue constructs of clinical relevance. In this work, we present the use of an impedance spectroscopy technique for the real-time measurement of the dielectric properties of skeletal myoblast cell cultures. The processes involved in the growth and differentiation of these cell cultures in skeletal muscle are studied. A circuit based on the oscillation-based test technique was used, avoiding the use of high-performance circuitry or external input signals. The effect of electrical pulse stimulation applied to cell cultures was also studied. The technique proved useful for monitoring in real-time the processes of cell growth and estimating the fill factor of muscular stem cells. Impedance spectroscopy was also useful to study the real-time monitoring of cell differentiation, obtaining different oscillation amplitude levels for differentiated and undifferentiated cell cultures. Finally, an electrical model was implemented to better understand the physical properties of the cell culture and control the tissue formation process.Spanish Government’s Ministerio de Ciencia, Innovación y Universidades, Plan Estatal 2017-2020 Retos- Proyectos I+D+I and FEDER RTI2018-093512-B-C2

Synthetic Studies in Phytochrome Chemistry

An account is given of the author’s several approaches to the synthesis of the parent chromophore of phytochrome (1), a protein-bound linear tetrapyrrole derivative that controls photomorphogenesis in higher plants. These studies culminated in enantioselective syntheses of both (2R)- and (2S)-phytochromobilin (4), as well as several 13C-labeled derivatives designed to probe the site of Z,E-isomerization during photoexcitation. When reacted in vitro, synthetic 2R-4 and recombinant-derived phytochrome apoprotein N-C produced a protein-bound chromophore with identical difference spectra to naturally occurring 1

Experiência de familiares no cuidado a adolescentes com diabetes mellitus tipo 1

http://dx.doi.org/10.5902/217976928074Objective: to know the experience of relatives in caring for adolescents with type 1 diabetes mellitus. Method: a qualitative, exploratory and descriptive study. It was carried out ten semi-structured interviews with families of adolescents with type 1 diabetes mellitus, interpreted from analysis of thematic content. Results: after data analysis, it revealed three categories: Knowledge of family about type 1 diabetes; Difficulties adolescents in adherence to treatment of diabetes type 1: the look of the family; and difficulties experienced by the family regarding need of care to the adolescents with type 1 diabetes. Conclusions: the experiences of families of adolescents with type 1 diabetes are permeated by concerns and difficulties. The offering of multidisciplinary care and investments in health care may help adolescents and families in the care of the disease.Objetivo: conocer la experiencia de los familiares en el cuidado de los adolescentes con diabetes mellitus tipo 1. Método: estudio cualitativo, exploratorio y descriptivo. Fueron realizadas diez entrevistas semiestructuradas con las familias de los adolescentes con diabetes mellitus tipo 1, interpretada por el análisis de contenido temático. Resultados: después del análisis de los datos se revelaron tres categorías: conocimiento de los familiares sobre diabetes mellitus tipo 1, las dificultades de los adolescentes en la adherencia al tratamiento de la enfermedad: la mirada de los familiares; y las dificultades experimentadas por los familiares acerca de la necesidad de cuidados a los adolescentes con diabetes tipo 1. Conclusiones: las experiencias de los familiares de los adolescentes con diabetes están permeadas por preocupaciones y dificultades. La oferta de la atención multidisciplinaria e inversiones en la asistencia de la salud puede ayudar los adolescentes y las familias en el cuidado de la enfermedad.http://dx.doi.org/10.5902/217976928074Objetivo: conhecer a experiência de familiares no cuidado a adolescentes com diabetes mellitus tipo 1. Método: estudo qualitativo, exploratório e descritivo. Realizaram-se dez entrevistas semiestruturadas com familiares de adolescentes com diabetes mellitus tipo 1, interpretadas a partir da análise de conteúdo temática. Resultados: após a análise dos dados, emergiram três categorias: conhecimento dos familiares sobre diabetes mellitus tipo 1; dificuldades do adolescente na adesão ao tratamento do diabetes mellitus tipo 1: o olhar dos familiares; e dificuldades vivenciadas pela família frente à necessidade de cuidado ao adolescente com diabetes tipo 1. Conclusões: as experiências dos familiares de adolescentes com diabetes são permeadas por preocupações e dificuldades. O oferecimento da assistência multiprofissional e os investimentos na assistência à saúde podem auxiliar adolescentes e familiares nos cuidados com a doença

Hormonal Influence on Fat Synthesis in Cattle

The ability of adenosine, insulin and human acylation-stimulating protein to modify fat synthesis was determined using cultures of fat tissue from steers. Adenosine did not influence fat synthesis. However, acylation stimulating protein and insulin promoted fat synthesis. These observations, coupled with knowledge of how fat synthesis is regulated in other species, justify investigation of whether cattle synthesize acylation-stimulating protein, and how this synthesis is regulated. An understanding of how acylation-stimulating protein production and action is regulated should expose potential places for intervention to manipulate fat synthesis in cattle

Acylation Stimulating Protein: A Potential Regulator of Fat Synthesis

The long term goal of this project is to understand the molecular mechanisms controlling fat synthesis. These experiments indicate that acylation stimulating protein (ASP) can stimulate the incorporation of fatty acids into lipid in cultured adipose tissue. This finding justifies a future effort to determine if manipulation of ASP can modify fat deposition

Purification and characterization of acylation stimulating protein from porcine serum

A method for purifying acylation stimulating protein (ASP) from porcine serum is described. The mRNA encoding ASP was cloned by reverse transcriptase-polymerase chain reaction which predicted a 76 residue peptide. Based on this sequence, we generated antisera to a C-terminal peptide (ASP1–20) which aided ASP purification. Identity of the purified protein was verified by N-terminal sequencing. The molecular mass of porcine ASP is 8926. Porcine ASP stimulated esterification of fatty acid into triacylglycerol in cultured human cells with potency similar to that of human ASP (twofold at 5 μM). Based on this evidence that ASP exists in porcine blood, and that it has acylation stimulating activity, we propose that ASP may play a role in regulation of energy storage in adipose tissue in the pig

Histological tissue structure alterations resulting from Staphylococcus aureus intramammary infection in heifer mammary glands hormonally induced to rapidly grow and develop

ABSTRACT: Intramammary infections (IMI) are common in nonlactating dairy cattle and are expected to impair mammary growth and development and reduce future milk production. The objective of this study was to histologically evaluate how IMI alter tissue structure in growing and developing heifer mammary glands. A total of 18 nonpregnant, nonlactating heifers between 11 and 14 mo of age were used in the present study. Heifers received daily supraphysiological injections of estradiol and progesterone for 14 d to stimulate rapid mammary growth and development. One-quarter of each heifer was subsequently infused with Staphylococcus aureus (CHALL) while a second quarter served as an uninfected control (UNINF). Heifers were randomly selected and euthanized either the last day of hormonal injections to observe IMI effects on mammary gland growth (GRO), or 13 d post-injections, to observe IMI effects on mammary development (DEV). Mammary tissues were collected from the center and edge parenchymal regions of each mammary gland for morphometric tissue area evaluation. For GRO tissues, CHALL quarters had less epithelial tissue area and marginally more intralobular stroma tissue area than UNINF quarters. Tissue areas occupied by luminal space, extralobular stroma, adipose, and lobular tissue were similar. For DEV tissues, area occupied by epithelium, luminal space, intralobular stroma, and extralobular stroma did not differ between quarter treatments, but UNINF quarters had more adipose tissue area and marginally less lobular area than CHALL quarters. Results indicate that IMI in growing and developing mammary glands reduces mammary epithelial growth and alters mammary gland development by impairing epithelial branching into the mammary fat pad. Taken together, these tissue changes before calving may have adverse effects on milk production. Therefore, an important focus should be placed on improving udder health in replacement heifers through management strategies that mitigate the deleterious effects of IMI and promote the positive development of the mammary gland