TGR5 contributes to glucoregulatory improvements after vertical sleeve gastrectomy in mice.

OBJECTIVE: Vertical sleeve gastrectomy (VSG) produces high rates of type 2 diabetes remission; however, the mechanisms responsible remain incompletely defined. VSG increases circulating bile acid concentrations and bile acid signalling through TGR5 improves glucose homeostasis. Therefore, we investigated the role of TGR5 signalling in mediating the glucoregulatory benefits of VSG. DESIGN: VSG or sham surgery was performed in high-fat-fed male Tgr5+/+ (wild type) and Tgr5-/- (knockout) littermates. Sham-operated mice were fed ad libitum or food restricted to match their body weight to VSG-operated mice. Body weight, food intake, energy expenditure, insulin signalling and circulating bile acid profiles were measured and oral glucose tolerance testing, islet immunohistochemistry and gut microbial profiling were performed. RESULTS: VSG decreased food intake and body weight, increased energy expenditure and circulating bile acid concentrations, improved fasting glycaemia, glucose tolerance and glucose-stimulated insulin secretion, enhanced nutrient-stimulated glucagon-like peptide 1 secretion and produced favourable shifts in gut microbial populations in both genotypes. However, the body weight-independent improvements in fasting glycaemia, glucose tolerance, hepatic insulin signalling, hepatic inflammation and islet morphology after VSG were attenuated in Tgr5-/- relative to Tgr5+/+ mice. Furthermore, VSG produced metabolically favourable alterations in circulating bile acid profiles that were blunted in Tgr5-/- relative to Tgr5+/+ mice. TGR5-dependent regulation of hepatic Cyp8b1 expression may have contributed to TGR5-mediated shifts in the circulating bile acid pool after VSG. CONCLUSIONS: These results suggest that TGR5 contributes to the glucoregulatory benefits of VSG surgery by promoting metabolically favourable shifts in the circulating bile acid pool.

Maternal ileal interposition surgery confers metabolic improvements to offspring independent of effects on maternal body weight in UCD-T2DM rats.

BACKGROUND: Increasing numbers of people are undergoing bariatric surgery, of which approximately half are women in their childbearing years. However, information on the long-term effects of maternal bariatric surgery in their children is lacking. Furthermore, since bariatric surgery is performed to reduce body weight, clinical studies have not been able to differentiate between benefits to the child due to maternal body weight loss versus other maternal postoperative metabolic changes. Therefore, we used the University of California, Davis, type 2 diabetes mellitus (UCD-T2DM) rat model to test the hypothesis that maternal ileal interposition (IT) surgery would confer beneficial metabolic effects in offspring, independent of effects on maternal body weight. METHODS: IT surgery was performed on 2-month-old prediabetic female UCD-T2DM rats. Females were bred 3 weeks after surgery, and male pups were studied longitudinally. RESULTS: Maternal IT surgery resulted in decreased body weight in offspring compared with sham offspring (P < 0.05). IT offspring exhibited improvements of glucose-stimulated insulin secretion and nutrient-stimulated glucagon-like peptide-2 (GLP-2) secretion (P < 0.05). Fasting plasma unconjugated bile acid concentrations were 4-fold lower in IT offspring compared with sham offspring at two months of age (P < 0.001). CONCLUSIONS: Overall, maternal IT surgery confers modest improvements of body weight and improves insulin secretion and nutrient-stimulated GLP-2 secretion in offspring in the UCD-T2DM rat model of type 2 diabetes, indicating that this is a useful model for investigating the weight-independent metabolic effects of maternal bariatric surgery.

Bile-acid-mediated decrease in endoplasmic reticulum stress: a potential contributor to the metabolic benefits of ileal interposition surgery in UCD-T2DM rats.

Post-operative increases in circulating bile acids have been suggested to contribute to the metabolic benefits of bariatric surgery; however, their mechanistic contributions remain undefined. We have previously reported that ileal interposition (IT) surgery delays the onset of type 2 diabetes in UCD-T2DM rats and increases circulating bile acids, independently of effects on energy intake or body weight. Therefore, we investigated potential mechanisms by which post-operative increases in circulating bile acids improve glucose homeostasis after IT surgery. IT, sham or no surgery was performed on 2-month-old weight-matched male UCD-T2DM rats. Animals underwent an oral fat tolerance test (OFTT) and serial oral glucose tolerance tests (OGTT). Tissues were collected at 1.5 and 4.5 months after surgery. Cell culture models were used to investigate interactions between bile acids and ER stress. IT-operated animals exhibited marked improvements in glucose and lipid metabolism, with concurrent increases in postprandial glucagon-like peptide-1 (GLP-1) secretion during the OFTT and OGTTs, independently of food intake and body weight. Measurement of circulating bile acid profiles revealed increases in circulating total bile acids in IT-operated animals, with a preferential increase in circulating cholic acid concentrations. Gut microbial populations were assessed as potential contributors to the increases in circulating bile acid concentrations, which revealed proportional increases in Gammaproteobacteria in IT-operated animals. Furthermore, IT surgery decreased all three sub-arms of ER stress signaling in liver, adipose and pancreas tissues. Amelioration of ER stress coincided with improved insulin signaling and preservation of ß-cell mass in IT-operated animals. Incubation of hepatocyte, adipocyte and ß-cell lines with cholic acid decreased ER stress. These results suggest that postoperative increases in circulating cholic acid concentration contribute to improvements in glucose homeostasis after IT surgery by ameliorating ER stress.

Vertical sleeve gastrectomy improves glucose and lipid metabolism and delays diabetes onset in UCD-T2DM rats.

Vertical sleeve gastrectomy (VSG) has gained interest as a low morbidity bariatric surgery, which is effective in producing weight loss and causing type 2 diabetes resolution. However, the efficacy of VSG to prevent the onset of type 2 diabetes has not been previously investigated. VSG or sham surgery was performed on 2-month-old prediabetic male University of California Davis-type 2 diabetes mellitus rats. Sham-operated animals were either sham-operated ad libitum fed (S-AL) or were weight-matched to VSG-operated animals (S-WM). Diabetes onset was determined by weekly nonfasting blood glucose measurements. Animals underwent oral glucose tolerance tests at 1 and 4 months after surgery and indirect calorimetry at 1.5 months after surgery. VSG surgery significantly delayed diabetes onset compared with both S-AL and S-WM animals. VSG-operated animals ate 23% less and weighed 20% less than S-AL. Energy expenditure did not differ between VSG-operated animals and controls. Results from the oral glucose tolerance tests demonstrate improved glucose tolerance and islet function in VSG-operated animals compared with S-AL and S-WM. Nutrient-stimulated glucagon-like peptide (GLP)-1, GLP-2, and peptide YY excursions were greater in VSG-operated animals. VSG surgery resulted in decreased fasting plasma insulin, ghrelin and lipid concentrations, and markedly higher fasting plasma adiponectin and bile acid concentrations, independent of body weight. Increases of circulating bile acid concentrations were due to selective increases of taurine-conjugated bile acids. Thus, VSG delays type 2 diabetes onset in the University of California Davis-type 2 diabetes mellitus rat, independent of body weight. This is potentially mediated by increases of circulating bile acids, adiponectin, and nutrient-stimulated GLP-1 secretion and decreased circulating ghrelin concentrations.

Varespladib (A-002), a secretory phospholipase A2 inhibitor, reduces atherosclerosis and aneurysm formation in ApoE-/- mice.

The family of secretory phospholipase A2 (sPLA2) enzymes has been associated with inflammatory diseases and tissue injury including atherosclerosis. A-001 is a novel inhibitor of sPLA2 enzymes discovered by structure-based drug design, and A-002 is the orally bioavailable prodrug currently in clinical development. A-001 inhibited human and mouse sPLA2 group IIA, V, and X enzymes with IC50 values in the low nM range. A-002 (1 mg/kg) led to high serum levels of A-001 and inhibited PLA2 activity in transgenic mice overexpressing human sPLA2 group IIA in C57BL/6J background. In addition, the effects of A-002 on atherosclerosis in 2 ApoE mouse models were evaluated using en face analysis. (1) In a high-fat diet model, A-002 (30 and 90 mg/kg twice a day for 16 weeks) reduced aortic atherosclerosis by 50% (P < 0.05). Plasma total cholesterol was decreased (P < 0.05) by 1 month and remained lowered throughout the study. (2) In an accelerated atherosclerosis model, with angiotensin II-induced aortic lesions and aneurysms, A-002 (30 mg/kg twice a day) reduced aortic atherosclerosis by approximately 40% (P < 0.05) and attenuated aneurysm formation (P = 0.0096). Thus, A-002 was effective at significantly decreasing total cholesterol, atherogenesis, and aneurysm formation in these 2 ApoE mouse models.

Investigation of the terminal P4 domain in a series of D-phenylglycinamide-based factor Xa inhibitors.

Several P4 domain derivatives of the general d-phenylglycinamide-based scaffold (2) were synthesized and evaluated for their ability to bind to the serine protease factor Xa. Some of the more potent compounds were evaluated for their anticoagulant effects in vitro. A select subset containing various P1 indole constructs was further evaluated for their pharmacokinetic properties after oral administration to rats.

Children's questions: a mechanism for cognitive development.

Preschoolers' questions may play an important role in cognitive development. When children encounter a problem with their current knowledge state (a gap in their knowledge, some ambiguity they do not know how to resolve, some inconsistency they have detected), asking a question allows them to get targeted information exactly when they need it. This information is available to them when they are particularly receptive to it, and because it comes as the result of their own disequilibrium, it may have depth of processing benefits. In that questions allow children to get information they need to move their knowledge structures closer to adult-like states, the ability to ask questions to gather needed information constitutes an efficient mechanism for cognitive development (referred to in this paper as the Information Requesting Mechanism [IRM]; this term is used because it includes question-asking and other information recruiting behaviors such as gestures, expressions, and vocalizations). However, the role of children's questions in their cognitive development has been largely overlooked. If questions are a force in cognitive development, the following must be true: (1) children must actually ask questions that gather information; (2) children must receive informative answers to their questions if they are able to be of use to cognitive development; (3) children must be motivated to get the information they request, rather than asking questions for other purposes such as attention; (4) the questions children ask must be relevant and of potential use to their cognitive development; (5) we must see evidence that children's questions help them in some way-that is, that they can ask questions for a purpose, and use the information they receive purposefully to successfully achieve some change of knowledge state. This monograph reports data on these points. Study 1 analyzed questions taken from four children's transcripts in the CHILDES database (age 1;2-5;1). This methodology allowed detailed, veridical analysis of every question asked by the children during their recording sessions. Results indicate that children ask many information-seeking questions and get informative answers. When they do not get an informative response, they keep asking; attention is not enough. Results also indicate that the content of children's questions parallel their conceptual advances, and shift within an exchange and over the course of development to reflect the learning process. So, these data suggest that the components of the IRM are in place and are used by children from very early in development, and the information they seek changes with time. Study 2 asked whether preverbal children who are not yet asking linguistic questions can recruit information via gestures, expressions, and vocalizations, in addition to further investigating the linguistic questions of older children. This study analyzed questions from a cross-sectional diary study, kept by 68 parents of their children's questions (aged 1;0-5;0). Also, this methodology allowed for data collection over a large number of children, a large range of situational contexts, and allows for the collection of low frequency, high-salience events. Results from Study 2 suggest that all of the components of the IRM are in place, and extends these findings down to younger, preverbal children who recruit information using gesture and vocalizations. Study 3 investigated the questions asked in one specific domain, biological knowledge, and examined the impact that different stimulus types have on children's questions. This study gathered data from 112 parent/child dyads (children aged 2, 3, and 4 years) walking through one of three zoos (one with real animals, one with drawings of animals, and one with three-dimensional replicas of animals), looking at the animals together. Results from this study also suggest that all of the components of the IRM are in place from the earliest age, further supporting the findings from Studies 1 and 2. In addition, while children still ask many nonbiological questions about the animals ("what is its name?"), biological information ("how do babies grow their bees?") is requested with much greater frequency in this study, although this need not necessarily be the case. Further, the nature of these questions suggests they may support the building of conceptual structures within the domain of biological knowledge, at a time just before the age when children make important conceptual changes in this area. Further, the type of stimulus materials used has an impact on the questions children ask; children are less likely to ask deep conceptual questions when looking at drawings or replicas of objects than when looking at the real thing. Finally, Study 4 examines the causal relation between children's questions and change in knowledge state by investigating whether or not children can ask questions in order to gain information that allows them to solve a problem. Sixty-seven 4-year-olds were asked to figure out which of two items were hidden in a box. Half of the children were allowed to ask questions to help them figure this out. Despite many ways in which they could fail to use questions correctly, children who were allowed to ask questions were significantly more likely to identify the object hidden in the box, an overt indication of their change in knowledge state. Further, children relied on their existing conceptual information about the objects to help generate disambiguating questions; even though they had a faster "dumb" method of disambiguating the objects via nonconceptual perceptual information ("is it purple?"), they were just as likely to generate questions that tapped into nonvisible conceptual information ("does it purr?"). These results suggest that children are capable of using their existing knowledge structures to generate questions that change their knowledge state in a way that allows them to productively solve a problem; they further suggest that tapping into existing conceptual knowledge to help process a current situation, and use that knowledge to generate appropriate questions, is an integral part of question asking. Together, the results of these four studies support the existence of the IRM as a way for children to learn about the world. Children ask information-seeking questions that are related in topic and structure to their cognitive development. Parents give answers to these questions, but when they do not, the children persist in asking for the information, suggesting that the goal of this behavior is to recruit needed information. The content of these questions shifts within exchanges and over the course of development in ways that reflect concept building. Finally, children generate questions efficiently in order to gather needed information, and then are able to use this information productively; they tap into their existing conceptual knowledge in order to do this. Thus, the ability to ask questions is a powerful tool that allows children to gather information they need in order to learn about the world and solve problems in it. Implications of this model for cognitive development are discussed.

Farnesoid X receptor agonist reduces serum asymmetric dimethylarginine levels through hepatic dimethylarginine dimethylaminohydrolase-1 gene regulation.

The farnesoid X receptor (FXR, NR1H4) is a bile acid-responsive nuclear receptor that plays critical roles in the transcriptional regulation genes involved in cholesterol, bile acid, triglyceride, and carbohydrate metabolism. By microarray analysis of hepatic genes from female Zucker diabetic fatty (ZDF) rats treated with the FXR agonist GW4064, we have identified dimethylarginine dimethylaminohydrolase-1 (DDAH1) as an FXR target gene. DDAH1 is a key catabolic enzyme of asymmetric dimethylarginine (ADMA), a major endogenous nitric-oxide synthase inhibitor. Sequence analysis of the DDAH1 gene reveals the presence of an FXR response element (FXRE) located 90 kb downstream of the transcription initiation site and within the first intron. Functional analysis of the putative FXRE demonstrated GW4064 dose-dependent transcriptional activation from the element, and we have demonstrated that the FXRE sequence binds the FXR-RXR heterodimer. In vivo administration of GW4064 to female ZDF rats promoted a dose-dependent and >6-fold increase in hepatic DDAH1 gene expression. The level of serum ADMA was reduced concomitantly. These findings provide a mechanism by which FXR may increase endothelium-derived nitric oxide levels through modulation of serum ADMA levels via direct regulation of hepatic DDAH1 gene expression. Thus, beneficial clinical outcomes of FXR agonist therapy may include prevention of atherosclerosis and improvement of the metabolic syndrome.

Spontaneous activation and signaling by overexpressed epidermal growth factor receptors in glioblastoma cells.

Overexpressed epidermal growth receptor factor receptors (EGFRs) are thought to contribute to the malignant phenotype of human glioblastomas (GBMs), but the mechanism is not well understood. We found that SKMG-3 cells, a rare GBM cell line that maintains EGFR gene amplification in vitro, produced high levels of EGFR protein. The cells also expressed the related receptors HER2/neu and HER4, but not HER3. Immunoblots and tryptic phosphopeptide maps showed that the SKMG-3 EGFRs were intact and functional and that a subset of these receptors were spontaneously autophosphorylated. EGF treatment stimulated phosphorylation of the EGFRs as well as the downstream effectors Erk, AKT1, stat3 and c-Cbl. Under minimal growth conditions, the unstimulated SKMG-3 cells contained constitutively phosphorylated Erk and AKTI but no detectable stat3 DNA-binding complexes. The EGFR kinase inhibitor PD158780 reduced the constitutive phosphorylation of the receptor and Erk but not that of AKT1. In contrast, inhibition of phosphatidylinositol-3-kinase (PI3K) blocked the constitutive phosphorylation of Erk and AKT-1 but not the EGFR. We conclude that the SKMG-3 cells represent the subset of GBMs with amplified EGFR genes that overexpress intact receptors. The results also suggest that in some GBMs, signals from overexpressed EGFRs contribute to the constitutive phosphorylation of Erk, but these signals may not required for the constitutive activation of PI3K or AKT1.