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Aims. Posttranslational modifications of histones and transcription factors regulate gene expression and are implicated in beta-cell failure and diabetes. We have recently shown that preserving H3K27 and H3K4 methylation using the lysine demethylase inhibitor GSK-J4 reduces cytokine-induced destruction of beta-cells and improves beta-cell function. Here, we investigate the therapeutic potential of GSK-J4 to prevent diabetes development and examine the importance of H3K4 methylation for islet function. Materials and Methods. We used two mouse models of diabetes to investigate the therapeutic potential of GSK-J4. To clarify the importance of H3K4 methylation, we characterized a mouse strain with knockout (KO) of the H3K4 demethylase KDM5B. Results. GSK-J4 administration failed to prevent the development of experimental diabetes induced by multiple low-dose streptozotocin or adoptive transfer of splenocytes from acutely diabetic NOD to NODscid mice. KDM5B-KO mice were growth retarded with altered body composition, had low IGF-1 levels, and exhibited reduced insulin secretion. Interestingly, despite secreting less insulin, KDM5B-KO mice were able to maintain normoglycemia following oral glucose tolerance test, likely via improved insulin sensitivity, as suggested by insulin tolerance testing and phosphorylation of proteins belonging to the insulin signaling pathway. When challenged with high-fat diet, KDM5B-deficient mice displayed similar weight gain and insulin sensitivity as wild-type mice. Conclusion. Our results show a novel role of KDM5B in metabolism, as KDM5B-KO mice display growth retardation and improved insulin sensitivity.

C4.4A is a modular glycolipid-anchored Ly6/uPAR/alpha-neurotoxin multidomain protein that exhibits a prominent membrane-associated expression in stratified squamous epithelia. C4.4A is also expressed in various solid cancer lesions, where high expression levels often are correlated to poor prognosis. Circumstantial evidence suggests a role for C4.4A in cell adhesion, migration, and invasion, but a well-defined biological function is currently unknown. In the present study, we have generated and characterized the first C4.4A-deficient mouse line to gain insight into the functional significance of C4.4A in normal physiology and cancer progression. The unchallenged C4.4A-deficient mice were viable, fertile, born in a normal Mendelian distribution and, surprisingly, displayed normal development of squamous epithelia. The C4.4A-deficient mice were, nonetheless, significantly lighter than littermate controls predominantly due to differences in fat mass. Congenital C4.4A deficiency delayed migration of keratinocytes enclosing incisional skin wounds in male mice. In chemically induced bladder carcinomas, C4.4A deficiency attenuated the incidence of invasive lesions despite having no effect on total tumour burden. This new C4.4A-deficient mouse line provides a useful platform for future studies on functional aspects of C4.4A in tumour cell invasion in vivo., C4.4A is a modular glycolipid-anchored Ly6/uPAR/alpha-neurotoxin multidomain protein that exhibits a prominent membrane-associated expression in stratified squamous epithelia. C4.4A is also expressed in various solid cancer lesions, where high expression levels often are correlated to poor prognosis. Circumstantial evidence suggests a role for C4.4A in cell adhesion, migration, and invasion, but a well-defined biological function is currently unknown. In the present study, we have generated and characterized the first C4.4A-deficient mouse line to gain insight into the functional significance of C4.4A in normal physiology and cancer progression. The unchallenged C4.4A-deficient mice were viable, fertile, born in a normal Mendelian distribution and, surprisingly, displayed normal development of squamous epithelia. The C4.4A-deficient mice were, nonetheless, significantly lighter than littermate controls predominantly due to differences in fat mass. Congenital C4.4A deficiency delayed migration of keratinocytes enclosing incisional skin wounds in male mice. In chemically induced bladder carcinomas, C4.4A deficiency attenuated the incidence of invasive lesions despite having no effect on total tumour burden. This new C4.4A-deficient mouse line provides a useful platform for future studies on functional aspects of C4.4A in tumour cell invasion in vivo.

Whey protein consumption reportedly alleviates parameters of the metabolic syndrome. Here, we investigated the effects of whey protein isolate (whey) in young mice fed a high-fat diet. We hypothesized that whey as the sole protein source reduced early weight gain associated with retarded growth and decreased concentration of insulin-like growth factor-1. Moreover, we hypothesized that these changes were explained by increased nitrogen loss via elevated urea production and/or increased energy expenditure. Male 5-week-old C57BL/6 mice were fed high-fat diets with the protein source being either whey, casein or a combination of both for 5 weeks. After 1, 3 or 5 weeks, respectively, the mice were subjected to a meal challenge with measurements of blood and urinary urea before and 1 and 3 h after eating a weighed meal of their respective diets. In a subset of mice, energy expenditure was measured by indirect calorimetry during the first week of dietary intervention. Observed exclusively during the first week of intervention, whey significantly reduced body length (P<.01) and weight gain (P<.001) correlating positively with plasma concentrations of insulin-like growth factor-1. The combination diet displayed intermediate results indicating an interactive effect. Urea production, urea cycle activity, food intake and energy expenditure were unaffected by protein source. In conclusion, whey decreased growth-related parameters exclusively during the first week of dietary intervention. The early effect of whey could not be explained by food intake, energy expenditure, urea production or urea cycle activity but was correlated with plasma levels of insulin-like growth factor-1.

Erythropoietin (EPO) ameliorates glucose metabolism through mechanisms not fully understood. In this study, we investigated the effect of EPO on glucose metabolism and insulin signaling in skeletal muscle. A 2-week EPO treatment of rats fed with a high-fat diet (HFD) improved fasting glucose levels and glucose tolerance, without altering total body weight or retroperitoneal fat mass. Concomitantly, EPO partially rescued insulin-stimulated AKT activation, reduced markers of oxidative stress, and restored heat-shock protein 72 expression in soleus muscles from HFD-fed rats. Incubation of skeletal muscle cell cultures with EPO failed to induce AKT phosphorylation and had no effect on glucose uptake or glycogen synthesis. We found that the EPO receptor gene was expressed in myotubes, but was undetectable in soleus. Together, our results indicate that EPO treatment improves glucose tolerance but does not directly activate the phosphorylation of AKT in muscle cells. We propose that the reduced systemic inflammation or oxidative stress that we observed after treatment with EPO could contribute to the improvement of whole-body glucose metabolism.

In order to characterize mechanisms responsible for fat accumulation we used a selectively bred obesity-prone (OP) and obesity-resistant (OR) rat model, where the rats were fed a Western diet for 76 days. Body composition was assessed by MRI scans and as expected the OP rats developed a higher degree of fat accumulation compared to OR rats. Indirect calorimetry showed that the OP rats had higher respiratory exchange ratio (RER) compared to OR rats indicating an impaired ability to oxidize fat. The OP rats had lower expression of carnitine palmitoyltransferase 1b in intra-abdominal fat, and higher expression of stearoyl-CoA desaturase 1 in subcutaneous fat compared to OR rats, which could explain the higher fat accumulation and RER values. Basal metabolic parameters were also examined in juvenile OP and OR rats before and during the introduction of the Western diet. Juvenile OP rats likewise had higher RER values indicating that this trait may be a primary and contributing factor to their obese phenotype. When the adult obese rats were exposed to the orexigenic and adipogenic hormone ghrelin, we observed increased RER values in both OP and OR rats, while OR rats were more sensitive to ghrelin's orexigenic effects as well as ghrelin-induced attenuation of activity and energy expenditure. Thus, increased fat accumulation characterizing obesity may be caused by impaired oxidative capacity due to decreased carnitine palmitoyltransferase 1b levels in the white adipose tissue, while ghrelin sensitivity did not seem to be a contributing factor.

Reactive oxygen species (ROS) contribute to target-cell damage in inflammatory and iron-overload diseases. Little is known about iron transport regulation during inflammatory attack. Through a combination of in vitro and in vivo studies, we show that the proinflammatory cytokine IL-1ß induces divalent metal transporter 1 (DMT1) expression correlating with increased ß cell iron content and ROS production. Iron chelation and siRNA and genetic knockdown of DMT1 expression reduce cytokine-induced ROS formation and cell death. Glucose-stimulated insulin secretion in the absence of cytokines in Dmt1 knockout islets is defective, highlighting a physiological role of iron and ROS in the regulation of insulin secretion. Dmt1 knockout mice are protected against multiple low-dose streptozotocin and high-fat diet-induced glucose intolerance, models of type 1 and type 2 diabetes, respectively. Thus, ß cells become prone to ROS-mediated inflammatory damage via aberrant cellular iron metabolism, a finding with potential general cellular implications.

L: -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.

Based on the conformationally constrained D-Trp-Phe-D-Trp (wFw) core of the prototype inverse agonist [D-Arg(1),D-Phe(5),D-Trp(7,9),Leu(11)]substance P, a series of novel, small, peptide-mimetic agonists for the ghrelin receptor were generated. By using various simple, ring-constrained spacers connecting the D-Trp-Phe-D-Trp motif with the important C-terminal carboxyamide group, 40 nm agonism potency was obtained and also in one case (wFw-Isn-NH(2), where Isn is isonipecotic acid) ~80% efficacy. However, in contrast to all previously reported ghrelin receptor agonists, the piperidine-constrained wFw-Isn-NH(2) was found to be a functionally biased agonist. Thus, wFw-Isn-NH(2) mediated potent and efficacious signaling through the Ga(q) and ERK1/2 signaling pathways, but in contrast to all previous ghrelin receptor agonists it did not signal through the serum response element, conceivably the Ga(12/13) pathway. The recognition pattern of wFw-Isn-NH(2) with the ghrelin receptor also differed significantly from that of all previously characterized unbiased agonists. Most importantly, wFw-Isn-NH(2) was not dependent on GluIII:09 (Glu3.33), which otherwise is an obligatory TM III anchor point residue for ghrelin agonists. Molecular modeling and docking experiments indicated that wFw-Isn-NH(2) binds in the classical agonist binding site between the extracellular segments of TMs III, VI, and VII, interacting closely with the aromatic cluster between TMs VI and VII, but that it does so in an opposite orientation as compared with, for example, the wFw peptide agonists. It is concluded that the novel peptide-mimetic ligand wFw-Isn-NH(2) is a biased ghrelin receptor agonist and that the selective signaling pattern presumably is due to its unique receptor recognition pattern lacking interaction with key residues especially in TM III.

GPR39, a constitutively active 7TM receptor important for glucose-induced insulin secretion and maturation of pancreatic ß-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 (Vo(2)) 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.

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.

The selective CB1 receptor antagonist rimonabant is a novel weight control agent. Although CB1 receptors and binding sites are present in both the rodent central and peripheral nervous systems, including the afferent vagus nerve, the role of gut afferents in mediating anorexia following CB1R blockade is still debated. In the present study we examined rimonabant-induced anorexia in male C57BL/6J mice with subdiaphragmatic vagotomy (VGX) as well as in male Sprague-Dawley rats subjected to either subdiaphragmatic vagal deafferentation (SDA) alone or in combination with a complete celiac-superior mesenteric ganglionectomy (CGX). Irrespective of the operational procedure, rimonabant (10mg/kg) effectively reduced standard chow as well as palatable diet (ensure) intake. In conclusion, the data clearly demonstrate that neither vagal gut afferents, nor gut afferents traveling via the sympathetic nervous system, are required for rimonabant to inhibit food intake leading to the hypothesis that centrally located CB1 receptors are the prime mediators of rimonabant-induced anorexia.

Udgivelsesdato: 2004-Jun, The ability of virus-specific CD8(+) T cells to produce cytokines was studied in mice infected with lymphocytic choriomeningitis virus and vesicular stomatitis virus. Intracellular staining was used to visualize cytokine-producing CD8(+) and CD4(+) T cells. Overall, virus-specific CD8(+) T cells produce a similar range of cytokines (IFN-gamma, TNF-alpha, IL-2, GM-CSF, RANTES, MIP-1alpha and MIP-1beta) as CD4(+) T cells, but the relative distribution of cytokine-producing subsets is different. Moreover, cytokine-producing CD8(+) T cells were found to dominate numerically at all time-points tested. Co-staining for more than one cytokine revealed that while all cytokine-producing CD8(+) T cells synthesized IFN-gamma, additional cytokines were produced by partly overlapping subsets of this population. The frequency of cells producing more than one cytokine was higher in a tertiary site (peritoneum) and generally increased with transition into the memory phase; however, GM-CSF producing cells were only present transiently. Concerning factors predicted to influence the distribution of cytokine-producing subsets, IFN-gamma and IL-12 did not play a role, nor was extensive virus replication essential. Notably, regarding the heterogeneity in cytokine production by individual cells with similar epitope specificity, variation in TCR avidity was not the cause, since in vivo-activated TCR transgene-expressing cells were as heterogeneous in cytokine expression as polyclonal cells specific for the same epitope.