Andreas N. Madsen

Long-term characterization of the diet-induced obese and diet-resistant rat model: a polygenetic rat model mimicking the human obesity syndrome.

The availability of useful animal models reflecting the human obesity syndrome is crucial in the search for novel compounds for the pharmacological treatment of obesity. In the current study, we have performed an extensive characterization of the obesity syndrome in a polygenetic animal model, namely the selectively bred diet-induced obese (DIO) and diet-resistant (DR) rat strains. We show that they constitute useful models of the human obesity syndrome. DIO and DR rats were fed either a high-energy (HE) or a standard chow (Chow) diet from weaning to 9 months of age. Metabolic characterization including blood biochemistry and glucose homeostasis was examined at 2, 3, 6, and 9 months of age. Furthermore, in 6-month-old HE-fed DIO rats, the anti-obesity effects of liraglutide and sibutramine were examined in a 28-day study. Only HE-fed DIO rats developed visceral obesity, hyperleptinemia, hyperinsulinemia, and dyslipidemia, and showed a worsening of glucose tolerance over time. In line with the hyperlipidemic profile, a severe hepatic fat infiltration was observed in DIO rats at 6 months of age. The effects of liraglutide and sibutramine were tested in 6-month-old DIO rats. Both compounds effectively reduced food intake and body weight in DIO rats. Liraglutide furthermore improved glucose tolerance when compared with sibutramine. Our data highlights the usefulness of a polygenetic animal model for screening of compounds affecting food intake, body weight, and glucose homeostasis. Furthermore, the results underscore the effectiveness of GLP-1 mimetics both as anti-diabetes and anti-obesity agents.

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.

Dietary fat decreases intestinal levels of the anorectic lipids through a fat sensor

This study was undertaken to investigate the link between dietary fat content and intestinal levels of anorectic AŁacylethanolamines (NAEs), including oleoyleth‐anolamide (OEA), palmitoylethanolamide (PEA), and lino‐leoylethanolamide (LEA). Male rats were fed high‐fat diets (HFDs) with variable percentages of fat [20–45% of total energy (E%)] for 1–7 d;afterward, the jejunums were isolated, and jejunal NAE levels were measured by liquid‐chromatography mass spectrometry. Enzyme activities and mRNA expression levels were measured for two synthesizing enzymes, Λ‐acylphosphatidylethanolamine‐specific phospholipase D (NAPE‐PLD) and glycerophos‐phodiesterase (GDE1), and one degrading enzyme, fatty acid amide hydrolase (FAAH). We found a dose‐response relation between the quantity/percentage of dietary fat, irrespective of the energy density, and the reduction of intestinal levels of OEA, PEA, and LEA. The reductions were present afterldof 45E% HFD. LEA, the major NAE species, was shown to have an anorectic potency slightly less than that of OEA but higher than PEA. Regulation at the enzyme level seems not to explain the changes in NAE levels. The results suggest the presence of a fat sensor, mediating the reduced intestinal NAE levels. The intestinal NAE levels are reduced in a dose‐ and time‐dependent manner in response to dietary fat intake, and this may contribute to the well‐known hyperphagic effect of HFDs.—Diep, T. A., Madsen, A. N., Holst, B., Kristiansen, M. M., Wellner, N., Hansen, S. H., Hansen, H. S. Dietary fat decreases intestinal levels of the anorectic lipids through a fat sensor. FASEB J. 25, 765–774 (2011). www.fasebj.org

Long-Acting Lipidated Analogue of Human Pancreatic Polypeptide Is Slowly Released into Circulation

The main disadvantages of peptide pharmaceuticals are their rapid degradation and excretion, their low hydrophilicity, and low shelf lifes. These bottlenecks can be circumvented by acylation with fatty acids (lipidation) or polyethylene glycol (PEGylation). Here, we describe the modification of a human pancreatic polypeptide analogue specific for the human (h)Y(2) and hY(4) receptor with PEGs of different size and palmitic acid. Receptor specificity was demonstrated by competitive binding studies. Modifications had only a small influence on binding affinities and no influence on secondary structure. Both modifications improved pharmacokinetic properties of the hPP analogue in vivo and in vitro, however, lipidation showed a greater resistance to degradation and excretion than PEGylation. Furthermore, the lipidated peptide is taken up and degraded solely by the liver but not the kidneys. Lipidation resulted in prolonged action of the hPP analogue in respect of reducing food intake in mice after subcutaneous administration. Therefore, the lipidated hPP analogue could constitute a potential new therapeutic agent against obesity.

Neurotensin Is Coexpressed, Coreleased, and Acts Together With GLP-1 and PYY in Enteroendocrine Control of Metabolism.

The 2 gut hormones glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) are well known to be coexpressed, costored, and released together to coact in the control of key metabolic target organs. However, recently, it became clear that several other gut hormones can be coexpressed in the intestinal-specific lineage of enteroendocrine cells. Here, we focus on the anatomical and functional consequences of the coexpression of neurotensin with GLP-1 and PYY in the distal small intestine. Fluorescence-activated cell sorting analysis, laser capture, and triple staining demonstrated that GLP-1 cells in the crypts become increasingly multihormonal, ie, coexpressing PYY and neurotensin as they move up the villus. Proglucagon promoter and pertussis toxin receptor-driven cell ablation and reappearance studies indicated that although all the cells die, the GLP-1 cells reappear more quickly than PYY- and neurotensin-positive cells. High-resolution confocal fluorescence microscopy demonstrated that neurotensin is stored in secretory granules distinct from GLP-1 and PYY storing granules. Nevertheless, the 3 peptides were cosecreted from both perfused small intestines and colonic crypt cultures in response to a series of metabolite, neuropeptide, and hormonal stimuli. Importantly, neurotensin acts synergistically, ie, more than additively together with GLP-1 and PYY to decrease palatable food intake and inhibit gastric emptying, but affects glucose homeostasis in a more complex manner. Thus, neurotensin is a major gut hormone deeply integrated with GLP-1 and PYY, which should be taken into account when exploiting the enteroendocrine regulation of metabolism pharmacologically.

Functionally biased signalling properties of 7TM receptors - opportunities for drug development for the ghrelin receptor.

The ghrelin receptor is a 7 transmembrane (7TM) receptor involved in a variety of physiological functions including growth hormone secretion, increased food intake and fat accumulation as well as modulation of reward and cognitive functions. Because of its important role in metabolism and energy expenditure, the ghrelin receptor has become an important therapeutic target for drug design and the development of anti‐obesity compounds. However, none of the compounds developed so far have been approved for commercial use. Interestingly, the ghrelin receptor is able to signal through several different signalling pathways including Gαq, Gαi/o, Gα12/13 and arrestin recruitment. These multiple signalling pathways allow for functionally biased signalling, where one signalling pathway may be favoured over another either by selective ligands or through mutations in the receptor. In the present review, we have described how ligands and mutations in the 7TM receptor may bias the receptors to favour either one G‐protein over another or to promote G‐protein independent signalling pathways rather than G–protein‐dependent pathways. For the ghrelin receptor, both agonist and inverse agonists have been demonstrated to signal more strongly through the Gαq‐coupled pathway than the Gα12/13‐coupled pathway. Similarly a ligand that promotes Gαq coupling over Gαi coupling has been described and it has been suggested that several different active conformations of the receptor may exist dependent on the properties of the agonist. Importantly, ligands with such biased signalling properties may allow the development of drugs that selectively modulate only the therapeutically relevant physiological functions, thereby decreasing the risk of side effects.

PICK1 Deficiency Impairs Secretory Vesicle Biogenesis and Leads to Growth Retardation and Decreased Glucose Tolerance

Two lipid membrane sculpting BAR domain proteins, PICK1 and ICA69, play a key role early in the biogenesis of peptide hormone secretory vesicles and are critical for normal growth and metabolic homeostasis.

l -Arginine improves multiple physiological parameters in mice exposed to diet-induced metabolic disturbances

Abstractl-Arginine (l-Arg) is a conditionally essential amino acid and a natural constituent of dietary proteins. Studies in obese rats and type 2 diabetic humans have indicated that dietary supplementation with l-Arg can diminish gain in white adipose tissue (WAT) and improve insulin sensitivity. However, the effects of l-Arg on glucose homeostasis, body composition and energy metabolism remain unclear. In addition, no studies have, to our knowledge, examined whether l-Arg has beneficial effects as a dietary supplement in the mouse model. In the present study, we investigated the effects of l-Arg supplementation to male C57BL/6 mice on an array of physiological parameters. l-Arg supplemented mice were maintained on a low-protein diet and body composition, appetite regulation, glucose tolerance, insulin sensitivity and energy expenditure were evaluated. A significant reduction in epididymal WAT was observed in l-Arg supplemented mice compared with mice fed an isocaloric control diet. Surprisingly, the l-Arg supplemented animals were hyperphagic corresponding to a highly significant decrease in feed efficiency, as body weight developed in a similar pattern in both experimental groups. Glucose homeostasis experiments revealed a major effect of l-Arg supplementation on glucose tolerance and insulin sensitivity, interestingly, independent of a parallel regulation in whole-body adiposity. Increased l-Arg ingestion also raised energy expenditure; however, no concurrent effect on locomotor activity, substrate metabolism or expression of uncoupling proteins (UCP1 and UCP2) in adipose tissues was displayed. In conclusion, dietary l-Arg supplementation substantially affects an array of metabolic-associated parameters including a reduction in WAT, hyperphagia, improved insulin sensitivity and increased energy expenditure in mice fed a low-protein diet.

Interleukin-18 activates skeletal muscle AMPK and reduces weight gain and insulin resistance in mice

Circulating interleukin (IL)-18 is elevated in obesity, but paradoxically causes hypophagia. We hypothesized that IL-18 may attenuate high-fat diet (HFD)-induced insulin resistance by activating AMP-activated protein kinase (AMPK). We studied mice with a global deletion of the α-isoform of the IL-18 receptor (IL-18R−/−) fed a standard chow or HFD. We next performed gain-of-function experiments in skeletal muscle, in vitro, ex vivo, and in vivo. We show that IL-18 is implicated in metabolic homeostasis, inflammation, and insulin resistance via mechanisms involving the activation of AMPK in skeletal muscle. IL-18R−/− mice display increased weight gain, ectopic lipid deposition, inflammation, and reduced AMPK signaling in skeletal muscle. Treating myotubes or skeletal muscle strips with IL-18 activated AMPK and increased fat oxidation. Moreover, in vivo electroporation of IL-18 into skeletal muscle activated AMPK and concomitantly inhibited HFD-induced weight gain. In summary, IL-18 enhances AMPK signaling and lipid oxidation in skeletal muscle implicating IL-18 in metabolic homeostasis.

Regulation of T cell migration during viral infection: role of adhesion molecules and chemokines

T cell mediated immunity and in particular CD8+ T cells are pivotal for the control of most viral infections. T cells exclusively exert their antiviral effect through close cellular interaction with relevant virus-infected target cells in vivo. It is therefore imperative that efficient mechanisms exist, which will rapidly direct newly generated effector T cells to sites of viral replication. In the present report we have reviewed our present knowledge concerning the molecular interactions, which are important in targeting of effector CD8+ T cells to sites of viral infection.