Maria Rasmussen

In Vivo Characterization of High Basal Signaling from the Ghrelin Receptor

The receptor for the orexigenic peptide, ghrelin, is one of the most constitutively active 7TM receptors known, as demonstrated under in vitro conditions. Change in expression of a constitutively active receptor is associated with change in signaling independent of the endogenous ligand. In the following study, we found that the expression of the ghrelin receptor in the hypothalamus was up-regulated approximately 2-fold in rats both during 48-h fasting and by streptozotocin-induced hyperphagia. In a separate experiment, to probe for the effect of the high basal signaling of the ghrelin receptor in vivo, we used intracerebroventricular administration by osmotic pumps of a peptide [D-Arg(1), D-Phe(5), D-Trp(7,9), Leu(11)]-substance P. This peptide selectively displays inverse agonism at the ghrelin receptor as compared with an inactive control peptide with just a single amino acid substitution. Food intake and body weight were significantly decreased in the group of rats treated with the inverse agonist, as compared with the groups treated with the control peptide or the vehicle. In the hypothalamus, the expression of neuropeptide Y and uncoupling protein 2 was decreased by the inverse agonist. In a hypothalamic cell line that endogenously expresses the ghrelin receptor, we observed high basal activity of the cAMP response element binding protein, an important signaling transduction pathway for appetite regulation. The activation was further increased by ghrelin administration and decreased by administration of the inverse agonist. It is suggested that the high constitutive signaling activity is important for the in vivo function of the ghrelin receptor in the control of food intake and body weight.

The Na+/H+ exchanger, NHE1, differentially regulates mitogen-activated protein kinase subfamilies after osmotic shrinkage in Ehrlich Lettre Ascites cells.

Osmotic stress modulates mitogen activated protein kinase (MAPK) activities, leading to altered gene transcription and cell death/survival balance, however, the mechanisms involved are incompletely elucidated. Here, we show, using a combination of biochemical and molecular biology approaches, that three MAPKs exhibit unique interrelationships with the Na+/H+ exchanger, NHE1, after osmotic cell shrinkage: Extracellular Signal Regulated Kinase (ERK1/2) is inhibited in an NHE1-dependent, pHi-independent manner, c-Jun N-terminal kinase (JNK1/2) is stimulated, in part through NHE1-mediated intracellular alkalinization, and p38 MAPK is activated in an NHE1-independent manner, and contributes to NHE1 activation and ERK inhibition. Shrinkage-induced ERK1/2 inhibition was attenuated in Ehrlich Lettre Ascites cells by NHE1 inhibitors (EIPA, cariporide) or removal of extracellular Na+, and mimicked by human (h) NHE1 expression in cells lacking endogenous NHE1 activity. The effect of NHE1 on ERK1/2 was pHi-independent and upstream of MEK1/2. Shrinkage-activation of JNK1/2 was attenuated by EIPA, augmented by hNHE1 expression, and abolished in the presence of HCO3-. Basal JNK activity was augmented at alkaline pHi. Shrinkage-activation of p38 MAPK was NHE1-independent, and p38 MAPK inhibition (SB203580) attenuated NHE1 activation and ERK1/2 inhibition. Long-term shrinkage elicited caspase-3 activation and a loss of cell viability, which was augmented by ERK1/2 or JNK1/2 inhibition, and attenuated by p38 MAPK inhibition.

Deficiency of the GPR39 receptor is associated with obesity and altered adipocyte metabolism

GPR39, a constitutively active 7TM receptor important for glucose‐induced insulin secretion and maturation of pancreatic β‐cell function, is up‐regulated in adipose tissue on abstinence from food and chemically induced diabetes. In the present study, we investigated the effect of GPR39 deficiency on body weight and adipocyte metabolism. GPR39‐deficient mice were subjected to a high‐fat diet and body composition, glucose tolerance, insulin secretion, food intake, and energy expenditure were evaluated. The cell biology of adipocyte metabolism was studied on both mRNA and protein levels. A significant increase in body weight corresponding to a 2‐fold selective increase in fat mass was observed in GPR39‐deficient mice fed a high‐fat diet as compared with wild‐type littermate controls fed the same diet. The GPR39‐deficient animals had similar food intake but displayed almost eliminated diet‐induced thermogenesis, measured by the oxygen consumption rate (VO2) on change from normal to high‐fat diet. Analysis of the adipose tissue for lipolytic enzymes demonstrated decreased level of phosphorylated hormone‐sensitive lipase (HSL) and a decreased level of adipose triglyceride lipase (ATGL) by 35 and 60%, respectively, after food withdrawal in the GPR39‐deficient mice. Extracellular signal‐regulated kinases (ERK1/2), a signaling pathway known to be important for lipolysis, was decreased by 56% in the GPR39‐deficient mice. GPR39 deficiency is associated with increased fat accumulation on a high‐fat diet, conceivably due to decreased energy expenditure and adipocyte lipolytic activity.—Petersen, P. S., Jin, C., Madsen, A. N., Rasmussen, M., Kuhre, R. L. Egerod, K. L., Nielsen, L. B., Schwartz, T. W., Holst, B. Deficiency of the GPR39 receptor is associated with obesity and altered adipocyte metabolism. FASEB J. 25, 3803–3814 (2011). www.fasebj.org

Long-term intended and unintended experiences after Advanced Life Support training

AIM Highly structured simulation-based training (SBT) on managing emergency situations can have a significant effect on immediate satisfaction and learning. However, there are some indications of problems when applying learned skills to practice. The aim of this study was to identify long-term intended and unintended learner reactions, experiences and reflections after attending a simulation based Advanced Life Support (ALS) course. METHOD Semi-structured interviews were conducted by telephone with a purposive sample of prior ALS-course participants. A constructivist grounded theory approach was used to analyze the data. RESULTS Seventeen former participants were interviewed. The main themes related to context adaptation, communities of practice and to transfer of skills. Interviewees described challenges in adapting to the structured simulation setting and going back to the uncertain and unstructured clinical world. In part, a result of the several conflicting communities of practice - one being the ALS-community and the others relating to professional roles. Despite reporting transferring a more systematic approach to managing patients in emergency situations and during ward rounds, surgery, and in their teaching, participants also reported poor transfer in emergency situations where not all team members had the same ALS-structured approach. CONCLUSION The result from this study indicates that the efficiency dimension of ALS competence is taught well in ALS courses, but that the form and content of these highly structured/model courses are insufficient in training the innovative dimension of competence that is needed for transfer of skills in unstructured, emergency situations.

The effect of constructing versus solving virtual patient cases on transfer of learning : a randomized trial

The purpose of this study was to explore the effect of actively constructing virtual patient (VP) cases compared with solving VP cases on knowledge gains, skills transfer and time spent on cases. Forty-five fourth-year medical students were randomized to constructing (VP-construction, n = 23) or solving (VP-solving, n = 22) four cardiopulmonary VP cases. Whereas the VP-solving group solved the cases, the VP-construction group only received the final diagnosis and had to complete the history, physical findings, and lab results. After a week, participants completed a transfer test involving two standardized patients representing cardiopulmonary cases. Performances on the transfer test were video-recorded and assessed by two blinded raters using the Reporter, Interpreter, Manager, Educator (RIME) framework. Thirty-nine participants completed the transfer test. The VP-construction group spent significantly more time on the VP cases compared with the VP-solving group, p = 0.002. There were no significant differences in RIME scores between the VP-construction group and VP-solving group, p = 0.54.In conclusion, engaging novice students in active VP case construction may be more time consuming than solving VP cases, without resulting in superior skills transfer.

Cupriavidus pinatubonensis AEO106 deals with copper-induced oxidative stress before engaging in biodegradation of the herbicide 4-chloro-2-methylphenoxyacetic acid

BackgroundMicrobial degradation of phenoxy acid (PA) herbicides in agricultural soils is important to minimize herbicide leaching to groundwater reservoirs. Degradation may, however, be hampered by exposure of the degrader bacteria to toxic metals as copper (Cu) in the soil environment. Exposure to Cu leads to accumulation of intracellular reactive oxygen species (ROS) in some bacteria, but it is not known how Cu-derived ROS and an ensuing oxidative stress affect the degradation of PA herbicides. Based on the previously proposed paradigm that bacteria deal with environmental stress before they engage in biodegradation, we studied how the degradation of the PA herbicide 2-methyl-4-chlorophenoxyacetic acid (MCPA) by the model PA degrader Cupriavidus pinatubonensis AEO106 was affected by Cu exposure.ResultsExposure of C. pinatubonensis in batch culture to sublethal concentrations of Cu increased accumulation of ROS measured by the oxidant sensing probe 2,7-dichlorodihydrofluorescein diacetate and flow cytometry, and resulted in upregulation of a gene encoding a protein belong to the Ohr/OsmC protein family. The ohr/osmC gene was also highly induced by H2O2 exposure suggesting that it is involved in the oxidative stress response in C. pinatubonensis. The increased ROS accumulation and increased expression of the oxidative stress defense coincided with a delay in the catabolic performance, since both expression of the catabolic tfdA gene and MCPA mineralization were delayed compared to unexposed control cells.ConclusionsThe current study suggests that Cu-induced ROS accumulation in C. pinatubonensis activates a stress response involving the product of the ohr/osmC gene. Further, the stress response is launched before induction of the catabolic tfdA gene and mineralization occurs.