Karolina Ploj

Opposing Effects of Adiponectin Receptors 1 and 2 on Energy Metabolism

The adipocyte-derived hormone adiponectin regulates glucose and lipid metabolism and influences the risk for developing obesity, type 2 diabetes, and cardiovascular disease. Adiponectin binds to two different seven-transmembrane domain receptors termed AdipoR1 and AdipoR2. To study the physiological importance of these receptors, AdipoR1 gene knockout mice (AdipoR1−/−) and AdipoR2 gene knockout mice (AdipoR2−/−) were generated. AdipoR1−/− mice showed increased adiposity associated with decreased glucose tolerance, spontaneous locomotor activity, and energy expenditure. However, AdipoR2−/− mice were lean and resistant to high-fat diet–induced obesity associated with improved glucose tolerance and higher spontaneous locomotor activity and energy expenditure and reduced plasma cholesterol levels. Thus, AdipoR1 and AdipoR2 are clearly involved in energy metabolism but have opposing effects.

Intestinal, adipose, and liver inflammation in diet-induced obese mice

Chronic inflammation and increased visceral adipose tissue (VAT) are key elements of the metabolic syndrome. Both are considered to play a pathogenic role in the development of liver steatosis and insulin resistance. The aim of the present study was to investigate the hypothesis that an inflamed intestine, induced both by diet and chemical irritation, could induce persistent inflammation in VAT. Female C57BL/6JOlaHsd mice were used. In study I, groups of mice (n = 6 per group) were given an obesity-inducing cafeteria diet (diet-induced obesity) or regular chow only (control) for 14 weeks. In study II, colitis in mice (n = 8) was induced by 3% dextran sulfate sodium in tap water for 5 days followed by 21 days of tap water alone. Healthy control mice (n = 8) had tap water only. At the end of the studies, all mice were killed; and blood and tissues were sampled and processed for analysis. Body weight of diet-induced obese mice was greatly increased, with evidence of systemic inflammation, insulin resistance, and liver steatosis. Tissue inflammation indexed by proinflammatory cytokine expression was recorded in liver, mesenteric fat, and proximal colon/distal ileum, but not in subcutaneous or perigonadal fat. In dextran sulfate sodium-induced colitis mice, mesenteric fat was even more inflamed than the colon, whereas a much milder inflammation was seen in liver and subcutaneous fat. The studies showed both diet- and colitis-initiated inflammation in mesenteric fat. Fat depots contiguous with intestine and their capacity for exaggerated inflammatory responses to conditions of impaired gut barrier function may account for the particularly pathogenic role of VAT in obesity-induced metabolic disorders.

Discovery of (3-(4-(2-Oxa-6-azaspiro[3.3]heptan-6-ylmethyl)phenoxy)azetidin-1-yl)(5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-yl)methanone (AZD1979), a Melanin Concentrating Hormone Receptor 1 (MCHr1) Antagonist with Favorable Physicochemical Properties

A novel series of melanin concentrating hormone receptor 1 (MCHr1) antagonists were the starting point for a drug discovery program that culminated in the discovery of 103 (AZD1979). The lead optimization program was conducted with a focus on reducing lipophilicity and understanding the physicochemical properties governing CNS exposure and undesired off-target pharmacology such as hERG interactions. An integrated approach was taken where the key assay was ex vivo receptor occupancy in mice. The candidate compound 103 displayed appropriate lipophilicity for a CNS indication and showed excellent permeability with no efflux. Preclinical GLP toxicology and safety pharmacology studies were without major findings and 103 was taken into clinical trials.

Central NMU signaling in body weight and energy balance regulation: evidence from NMUR2 deletion and chronic central NMU treatment in mice

To investigate the role of the central neuromedin U (NMU) signaling system in body weight and energy balance regulation, we examined the effects of long-term intracerebroventricular (icv) infusion of NMU in C57Bl/6 mice and in mice lacking the gene encoding NMU receptor 2. In diet-induced obese male and female C57BL/6 mice, icv infusion of NMU (8 microg x day(-1) x mouse(-1)) for 7 days decreased body weight and total energy intake compared with vehicle treatment. However, these parameters were unaffected by NMU treatment in lean male and female C57BL/6 mice fed a standard diet. In addition, female (but not male) NMUR2-null mice had increased body weight and body fat mass when fed a high-fat diet but lacked a clear body weight phenotype when fed a standard diet compared with wild-type littermates. Furthermore, female (but not male) NMUR2-null mice fed a high-fat diet were protected from central NMU-induced body weight loss compared with littermate wild-type mice. Thus, we provide the first evidence that long-term central NMU treatment reduces body weight, food intake, and adiposity and that central NMUR2 signaling is required for these effects in female but not male mice.

The metabotropic glutamate mGluR5 receptor agonist CHPG stimulates food intake

The metabotropic glutamate receptor 5 (mGluR5) has been suggested to modulate energy balance. For example, mGluR5 antagonists inhibit food intake in rodents and mGluR5 knockout mice resist diet-induced obesity. However, nonspecific effects can reduce food intake. Thus, to further support the role of mGluR5 in feeding behaviour, we evaluated if the mGluR5 agonist (R,S)-2-chloro-5-hydroxyphenylglycine (CHPG) would induce the opposite effect, i.e. increased food intake in rats. Intracerebroventricularly injected CHPG (0.5–1.5 μmol) induced a dose-dependent stimulation of food intake (349% increase at 2 h with 1.5 μmol). The mGluR5 antagonist 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]-pyridine (10 mg/kg intraperitoneally) reduced 24 h food intake, without altering CHPG-induced feeding. These findings further support a physiologically relevant role of mGluR5 in appetite regulation.

Effects of a novel potent melanin-concentrating hormone receptor 1 antagonist, AZD1979, on body weight homeostasis in mice and dogs.

Melanin‐concentrating hormone (MCH) is an orexigen, and while rodents express one MCH receptor (MCH1 receptor), humans, non‐human primates and dogs express two MCH receptors (MCH1 and MCH2). MCH1 receptor antagonists have been developed for the treatment of obesity and lower body weight in rodents. However, the mechanisms for the body weight loss and whether MCH1 receptor antagonism can lower body weight in species expressing both MCH receptors are not fully understood.

In vivo imaging of lipid storage and regression in diet-induced obesity during nutrition manipulation.

Changes in adipose tissue distribution and ectopic fat storage in, liver and skeletal muscle tissue impact whole body insulin sensitivity in both humans and experimental animals. Numerous mouse models of obesity, insulin resistance, and diabetes exist; however, current methods to assess mouse phenotypes commonly involve direct harvesting of the tissues of interest, precluding the possibility of repeated measurements in the same animal. In this study, we demonstrate that whole body 3-D imaging of body fat composition can be used to analyze distribution as well as redistribution of fat after intervention by repeated assessment of intrahepatocellular lipids (IHCL), intra-abdominal, subcutaneous, and total adipose tissue (IAT, SAT, and TAT) and brown adipose tissue (BAT). C57BL/6J mice fed a cafeteria diet for 16 wk were compared with mice fed standard chow for 16 wk and mice switched from café diet to standard chow after 12 wk. MRI determinations were made at 9 and 15 wk, and autopsy was performed at 16 wk. There was a strong correlation between MRI-calculated weights in vivo at 15 wk and measured weights at 16 wk ex vivo for IAT (r = 0.99), BAT (r = 0.93), and IHCL (r = 0.97). IHCL and plasma insulin increased steeply relative to body weight at body weights above 45 g. This study demonstrates that the use of 3-D imaging to assess body fat composition may allow substantial reductions in animal usage. The dietary interventions indicated that a marked metabolic deterioration occurred when the mice had gained a certain fat mass.

Refinement of habituation procedures in diet-induced obese mice.

Orogastric gavage, while a common method for delivering experimental substances in mice, has been shown to induce stress. To minimize the associated stress with this procedure, sham gavage prior to the start of experiment is a common method for habiutating mice. We investigated whether handling and restraint could replace sham treatment in the acclimatization protocol. Mice were either undisturbed, hand-restrained for 10 s or sham-gavaged daily for six days prior to eight days of twice daily gavage. The results showed that repetitive restraint and gavage had no differences in body weight after eight days of treatment compared with the body weights at the start of treatment, whereas animals left undisturbed lost significant weight once treatment began. These data suggest that procedure refinement by replacing sham treatment with hand restraint is sufficient to acclimatize mice to the stress associated with gavage.

Partial agonist activity of R3(BΔ23-27)R/I5 at RXFP3--investigation of in vivo and in vitro pharmacology.

Relaxin family peptide receptor 3 (RXFP3) is a G-protein coupled receptor mainly expressed in the brain and involved in appetite regulation. Previous studies in lean Wistar rats during the light phase have shown that the chimeric peptide R3(BΔ23-27)R/I5 suppresses food intake stimulated by an RXFP3 agonist, but has no effect on food intake when administered alone. We wanted to further investigate if R3(BΔ23-27)R/I5 on its own is able to antagonize the basal tone of the relaxin-3/RXFP3 system and therefore characterized the pharmacology of R3(BΔ23-27)R/I5 in vivo and in vitro. R3(BΔ23-27)R/I5 was intracerebroventricularly (ICV) injected in diet induced obese (DIO) Wistar rats and food intake was automatically measured during the dark phase when feeding drive is high. In our hands, R3(BΔ23-27)R/I5 alone did not have a significant effect on food intake during 24h following administration. Consistent with previous results, relaxin-3 stimulated food intake in satiated lean rats. R3(BΔ23-27)R/I5 was characterized in vitro using [(35)S]-GTPγS binding and cAMP assays, both assessing Gαi-protein mediated signalling, and dynamic mass redistribution (DMR) assays capturing the integrated cell response. R3(BΔ23-27)R/I5 showed partial agonist activity in all three functional assays. Thus, since R3(BΔ23-27)R/I5 displays partial RXFP3 agonist properties in vitro, further in vivo studies including additional tool compounds are needed to address if antagonizing relaxin-3/RXFP3 basal tone is a therapeutically relevant mechanism to regulate food intake and body weight.