Mathias Kranz

Adenosine activates brown adipose tissue and recruits beige adipocytes via A2A receptors

Brown adipose tissue (BAT) is specialized in energy expenditure, making it a potential target for anti-obesity therapies. Following exposure to cold, BAT is activated by the sympathetic nervous system with concomitant release of catecholamines and activation of β-adrenergic receptors. Because BAT therapies based on cold exposure or β-adrenergic agonists are clinically not feasible, alternative strategies must be explored. Purinergic co-transmission might be involved in sympathetic control of BAT and previous studies reported inhibitory effects of the purinergic transmitter adenosine in BAT from hamster or rat. However, the role of adenosine in human BAT is unknown. Here we show that adenosine activates human and murine brown adipocytes at low nanomolar concentrations. Adenosine is released in BAT during stimulation of sympathetic nerves as well as from brown adipocytes. The adenosine A2A receptor is the most abundant adenosine receptor in human and murine BAT. Pharmacological blockade or genetic loss of A2A receptors in mice causes a decrease in BAT-dependent thermogenesis, whereas treatment with A2A agonists significantly increases energy expenditure. Moreover, pharmacological stimulation of A2A receptors or injection of lentiviral vectors expressing the A2A receptor into white fat induces brown-like cells—so-called beige adipocytes. Importantly, mice fed a high-fat diet and treated with an A2A agonist are leaner with improved glucose tolerance. Taken together, our results demonstrate that adenosine–A2A signalling plays an unexpected physiological role in sympathetic BAT activation and protects mice from diet-induced obesity. Those findings reveal new possibilities for developing novel obesity therapies.

Gastric Bypass Surgery Recruits a Gut PPAR-α-Striatal D1R Pathway to Reduce Fat Appetite in Obese Rats

Bariatric surgery remains the single most effective long-term treatment modality for morbid obesity, achieved mainly by lowering caloric intake through as yet ill-defined mechanisms. Here we show in rats that Roux-en-Y gastric bypass (RYGB)-like rerouting of ingested fat mobilizes lower small intestine production of the fat-satiety molecule oleoylethanolamide (OEA). This was associated with vagus nerve-driven increases in dorsal striatal dopamine release. We also demonstrate that RYGB upregulates striatal dopamine 1 receptor (D1R) expression specifically under high-fat diet feeding conditions. Mechanistically, interfering with local OEA, vagal, and dorsal striatal D1R signaling negated the beneficial effects of RYGB on fat intake and preferences. These findings delineate a molecular/systems pathway through which bariatric surgery improves feeding behavior and may aid in the development of novel weight loss strategies that similarly modify brain reward circuits compromised in obesity.

Dissociation between brown adipose tissue 18F-FDG uptake and thermogenesis in uncoupling protein 1 deficient mice

18F-FDG PET imaging is routinely used to investigate brown adipose tissue (BAT) thermogenesis, which requires mitochondrial uncoupling protein 1 (UCP1). It remains uncertain, however, whether BAT 18F-FDG uptake is a reliable surrogate measure of UCP1-mediated heat production. Methods: UCP1 knockout (KO) and wild-type (WT) mice housed at thermoneutrality were treated with the selective β3 adrenergic receptor agonist CL 316, 243 and underwent metabolic cage, infrared thermal imaging and 18F-FDG PET/MRI experiments. Primary brown adipocytes were additionally examined for their bioenergetics by extracellular flux analysis as well as their uptake of 2-deoxy-3H-glucose. Results: In response to CL 316, 243 treatments, oxygen consumption, and BAT thermogenesis were diminished in UCP1 KO mice, but BAT 18F-FDG uptake was fully retained. Isolated UCP1 KO brown adipocytes exhibited defective induction of uncoupled respiration whereas their glycolytic flux and 2-deoxy-3H-glucose uptake rates were largely unaffected. Conclusion: Adrenergic stimulation can increase BAT 18F-FDG uptake independently of UCP1 thermogenic function.

Thyroid hormone status defines brown adipose tissue activity and browning of white adipose tissues in mice

The present study aimed to determine the effect of thyroid hormone dysfunction on brown adipose tissue activity and white adipose tissue browning in mice. Twenty randomized female C57BL/6NTac mice per treatment group housed at room temperature were rendered hypothyroid or hyperthyroid. In-vivo small animal 18F-FDG PET/MRI was performed to determine the effects of hypo- and hyperthyroidism on BAT mass and BAT activity. Ex-vivo14C-acetate loading assay and assessment of thermogenic gene and protein expression permitted analysis of oxidative and thermogenic capacities of WAT and BAT of eu-, hyper and hypothyroid mice. 18F-FDG PET/MRI revealed a lack of brown adipose tissue activity in hypothyroid mice, whereas hyperthyroid mice displayed increased BAT mass alongside enhanced 18F-FDG uptake. In white adipose tissue of both, hyper- and hypothyroid mice, we found a significant induction of thermogenic genes together with multilocular adipocytes expressing UCP1. Taken together, these results suggest that both the hyperthyroid and hypothyroid state stimulate WAT thermogenesis most likely as a consequence of enhanced adrenergic signaling or compensation for impaired BAT function, respectively.

Development of a High-Affinity PET Radioligand for Imaging Cannabinoid Subtype 2 Receptor

Cannabinoid receptors type 2 (CB2) represent a target with increasing importance for neuroimaging due to its upregulation under various pathological conditions. Encouraged by preliminary results obtained with [(11)C](Z)-N-(3-(2-methoxyethyl)-4,5-dimethylthiazol-2(3H)-ylidene)-2,2,3,3-tetramethyl-cyclopropanecarboxamide ([(11)C]A-836339, [(11)C]1) in a mouse model of acute neuroinflammation (induced by lipopolysaccharide, LPS), we designed a library of fluorinated analogues aiming for an [(18)F]-labeled radiotracer with improved CB2 binding affinity and selectivity. Compound (Z)-N-(3-(4-fluorobutyl)-4,5-dimethylthiazol-2(3H)-ylidene)-2,2,3,3-tetramethyl-cyclopropanecarboxamide (29) was selected as the ligand with the highest CB2 affinity (Ki = 0.39 nM) and selectivity over those of CB1 (factor of 1000). [(18)F]29 was prepared starting from the bromo precursor (53). Specific binding was shown in vitro, whereas fast metabolism was observed in vivo in CD-1 mice. Animal PET revealed a brain uptake comparable to that of [(11)C]1. In the LPS-treated mice, a 20-30% higher uptake in brain was found in comparison to that in nontreated mice (n = 3, P < 0.05).

Synthesis, 18F-Radiolabelling and Biological Characterization of Novel Fluoroalkylated Triazine Derivatives for in Vivo Imaging of Phosphodiesterase 2A in Brain via Positron Emission Tomography

Phosphodiesterase 2A (PDE2A) is highly and specifically expressed in particular brain regions that are affected by neurological disorders and in certain tumors. Development of a specific PDE2A radioligand would enable molecular imaging of the PDE2A protein via positron emission tomography (PET). Herein we report on the syntheses of three novel fluoroalkylated triazine derivatives (TA2–4) and on the evaluation of their effect on the enzymatic activity of human PDE2A. The most potent PDE2A inhibitors were 18F-radiolabelled ([18F]TA3 and [18F]TA4) and investigated regarding their potential as PET radioligands for imaging of PDE2A in mouse brain. In vitro autoradiography on rat brain displayed region-specific distribution of [18F]TA3 and [18F]TA4, which is consistent with the expression pattern of PDE2A protein. Metabolism studies of both [18F]TA3 and [18F]TA4 in mice showed a significant accumulation of two major radiometabolites of each radioligand in brain as investigated by micellar radio-chromatography. Small-animal PET/MR studies in mice using [18F]TA3 revealed a constantly increasing uptake of activity in the non-target region cerebellum, which may be caused by the accumulation of brain penetrating radiometabolites. Hence, [18F]TA3 and [18F]TA4 are exclusively suitable for in vitro investigation of PDE2A. Nevertheless, further structural modification of these promising radioligands might result in metabolically stable derivatives.

Development of a Novel Nonpeptidic 18F-Labeled Radiotracer for in Vivo Imaging of Oxytocin Receptors with Positron Emission Tomography

With the aim of imaging and quantification of oxytocin receptors (OTRs) in living brain using positron emission tomography (PET), we developed a (18)F-labeled small molecule radiotracer and investigated its in vivo pharmacokinetics in mice and pig. [(18)F]6b (KD = 12.3 nM) was radiolabeled by a two-step procedure using a microwave system with radiochemical yields of 26.9 ± 4.7%. Both organ distribution and small animal PET studies revealed limited brain uptake of [(18)F]6b in mouse (mean SUV of 0.04 at 30 min pi). Besides, significant radioactivity uptake in the pituitary gland was observed (SUV of 0.7 at 30 min pi). In a dynamic PET study in one piglet, we detected a higher uptake of [(18)F]6b in the olfactory bulb (SUV of 0.34 at 30 min pi) accompanied by a low uptake in the whole brain. In vitro autoradiographic studies on porcine brain sections indicated interaction of [(18)F]6b with several off-target receptors.

A novel thermoregulatory role for PDE10A in mouse and human adipocytes

Phosphodiesterase type 10A (PDE10A) is highly enriched in striatum and is under evaluation as a drug target for several psychiatric/neurodegenerative diseases. Preclinical studies implicate PDE10A in the regulation of energy homeostasis, but the mechanisms remain unclear. By utilizing small‐animal PET/MRI and the novel radioligand [18F]‐AQ28A, we found marked levels of PDE10A in interscapular brown adipose tissue (BAT) of mice. Pharmacological inactivation of PDE10A with the highly selective inhibitor MP‐10 recruited BAT and potentiated thermogenesis in vivo. In diet‐induced obese mice, chronic administration of MP‐10 caused weight loss associated with increased energy expenditure, browning of white adipose tissue, and improved insulin sensitivity. Analysis of human PET data further revealed marked levels of PDE10A in the supraclavicular region where brown/beige adipocytes are clustered in adults. Finally, the inhibition of PDE10A with MP‐10 stimulated thermogenic gene expression in human brown adipocytes and induced browning of human white adipocytes. Collectively, our findings highlight a novel thermoregulatory role for PDE10A in mouse and human adipocytes and promote PDE10A inhibitors as promising candidates for the treatment of obesity and diabetes.

Evaluation of the Enantiomer Specific Biokinetics and Radiation Doses of [18F]Fluspidine—A New Tracer in Clinical Translation for Imaging of σ1 Receptors

The enantiomers of [18F]fluspidine, recently developed for imaging of σ1 receptors, possess distinct pharmacokinetics facilitating their use in different clinical settings. To support their translational potential, we estimated the human radiation dose of (S)-(−)-[18F]fluspidine and (R)-(+)-[18F]fluspidine from ex vivo biodistribution and PET/MRI data in mice after extrapolation to the human scale. In addition, we validated the preclinical results by performing a first-in-human PET/CT study using (S)-(−)-[18F]fluspidine. Based on the respective time-activity curves, we calculated using OLINDA the particular organ doses (ODs) and effective doses (EDs). The ED values of (S)-(−)-[18F]fluspidine and (R)-(+)-[18F]fluspidine differed significantly with image-derived values obtained in mice with 12.9 μSv/MBq and 14.0 μSv/MBq (p < 0.025), respectively. A comparable ratio was estimated from the biodistribution data. In the human study, the ED of (S)-(−)-[18F]fluspidine was calculated as 21.0 μSv/MBq. Altogether, the ED values for both [18F]fluspidine enantiomers determined from the preclinical studies are comparable with other 18F-labeled PET imaging agents. In addition, the first-in-human study confirmed that the radiation risk of (S)-(−)-[18F]fluspidine imaging is within acceptable limits. However, as already shown for other PET tracers, the actual ED of (S)-(−)-[18F]fluspidine in humans was underestimated by preclinical imaging which needs to be considered in other first-in-human studies.

Internal Dose Assessment of (–)-18F-Flubatine, Comparing Animal Model Datasets of Mice and Piglets with First-in-Human Results

(−)-18F-flubatine is a promising tracer for neuroimaging of nicotinic acetylcholine receptors (nAChRs), subtype α4β2, using PET. Radiation doses after intravenous administration of the tracer in mice and piglets were assessed to determine the organ doses (ODs) and the effective dose (ED) to humans. The results were compared with subsequent clinical investigations in human volunteers. Methods: Twenty-seven female CD1 mice (weight ± SD, 28.2 ± 2.1 g) received intravenous injection of 0.75 ± 0.33 MBq of (−)-18F-flubatine. Up to 240 min after injection, 3 animals per time point were sacrificed and the organs harvested, weighed, and counted in a γ counter to determine mass and activity, respectively. Furthermore, whole-body PET scans of 5 female piglets (age ± SD, 44 ± 3 d; weight ± SD, 13.7 ± 1.7 kg) and 3 humans (2 men and 1 woman; age ± SD, 59.6 ± 3.9 y; weight ± SD, 74.3 ± 3.1 kg) were obtained up to 236 min (piglets) and 355 min (humans) after injection of 186.6 ± 7.4 and 353.7 ± 10.2 MBq of (−)-18F-flubatine, respectively, using a PET/CT scanner. The CT was used for delineation of the organs. Exponential curves were fitted to the time–activity-data, and time and mass scales were adapted to the human anatomy. The ODs were calculated using OLINDA/EXM (version 1.0); EDs were calculated with the tissue-weighting factors of ICRP103. Results: After the injection of (−)-18F-flubatine, there were no adverse or clinically detectable pharmacologic effects in any of the subjects. The highest activities after injection were found in the kidneys, urinary bladder, and liver. The urinary bladder receives the highest OD in all investigated species, followed by the kidneys and the liver for animals and humans, respectively. On the basis of mouse, piglet, and human kinetic data, the projected human ED of (−)-18F-flubatine was estimated to be 12.5 μSv/MBq in mice, 14.7 ± 0.7 μSv/MBq in piglets, and 23.4 ± 0.4 μSv/MBq in humans. Conclusion: As has been demonstrated for other PET radiotracers, preclinical (i.e., animal-derived) dosimetry underestimates the ED to humans, in the current case of (−)-18F-flubatine by 34%–44%.