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The response to prolonged fasting in hypothalamic serotonin transporter availability is blunted in obesity

Open AccessPublished:July 28, 2021DOI:https://doi.org/10.1016/j.metabol.2021.154839

      Highlights

      • Central serotonergic and dopaminergic systems control food intake and energy balance.
      • Serotonergic and dopaminergic responses to fasting have high inter-individual variation.
      • Fasting increases hypothalamic SERT availability in lean men, but not in obesity.
      • Fasting-induced free fatty acid (FFA) levels predict hypothalamic SERT availability.
      • Changes in FFAs and insulin together account for 44% of the striatal DAT response.

      Abstract

      Background and aims

      Serotonergic and dopaminergic systems in the brain are essential for homeostatic and reward-associated regulation of food intake and systemic energy metabolism. It is largely unknown how fasting influences these systems or if such effects are altered in humans with obesity. We therefore aimed to evaluate the effects of fasting on hypothalamic/thalamic serotonin transporter (SERT) and striatal dopamine transporter (DAT) availability in lean subjects and subjects with obesity.

      Methods

      In this randomized controlled cross-over trial, we assessed the effects of 12 vs 24 h of fasting on SERT and DAT availability in the hypothalamus/thalamus and striatum, respectively, using SPECT imaging in 10 lean men and 10 men with obesity.

      Results

      As compared with the 12-h fast, a 24-h fast increased hypothalamic SERT availability in lean men, but not in men with obesity. We observed high inter-individual variation in the effects of fasting on thalamic SERT and striatal DAT, with no differences between lean men and those with obesity. In all subjects, fasting-induced increases in circulating free fatty acid (FFA) concentrations were associated with an increase in hypothalamic SERT availability and a decrease in striatal DAT availability. Multiple regression analysis showed that changes in plasma insulin and FFAs together accounted for 44% of the observed variation in striatal DAT availability.

      Conclusion

      Lean men respond to prolonged fasting by increasing hypothalamic SERT availability, whereas this response is absent in men with obesity. Inter-individual differences in the adaptations of the cerebral serotonergic and dopaminergic systems to fasting may, in part, be explained by changes in peripheral metabolic signals of fasting, including FFAs and insulin.

      Keywords

      Abbreviations:

      123I-FP-CIT (123I-N-ω-fluoropropyl-2β-carboxymethoxy-3β-(4-iodophenyl)nortropane), ANOVA (analysis of variance), BMI (body mass index), BPND (binding potential), DAT (dopamine transporter), FFAR (free fatty acid receptor), FFA (free fatty acid), GPCR (G-protein-coupled receptor), IQR (interquartile range), MRI (magnetic resonance imaging), REE (resting energy expenditure), ROI (region-of-interest), RQ (respiratory quotient), SD (standard deviation), SERT (serotonin transporter), SPECT (single-photon emission computed tomography), T1w (T1-weighted)

      1. Introduction

      Obesity is defined as an excess of body fat and develops when long-term energy consumption exceeds energy expenditure [
      • Organization, W.H
      Factsheet: obesity and overweight.
      ]. Over the last decades, the increased availability of affordable, energy-dense food has paralleled an increase in average food consumption in many countries, and this is believed to be the main driver of the current obesity pandemic [
      • Swinburn B.A.
      • et al.
      The global obesity pandemic: shaped by global drivers and local environments.
      ,
      • Bluher M.
      Obesity: global epidemiology and pathogenesis.
      ]. On an individual level, the brain regulates many aspects of eating behavior and energy balance. Disturbances in the central regulation of food intake are hypothesized to underlie a pattern of eating behaviors in individuals with obesity that is characterized by ongoing caloric intake despite systemic energy surplus [
      • Berthoud H.R.
      • Munzberg H.
      • Morrison C.D.
      Blaming the brain for obesity: integration of hedonic and homeostatic mechanisms.
      ].
      Two important systems in the regulation of eating behavior by the brain are the homeostatic system and the reward system. The homeostatic system's primary function is to maintain energy homeostasis by matching the amount of calories consumed to the amount of calories expended [
      • Schwartz M.W.
      • et al.
      Central nervous system control of food intake.
      ]. The hypothalamus and brainstem are key brain regions for the homeostatic regulation of food intake [
      • Brobeck J.R.
      Mechanism of the development of obesity in animals with hypothalamic lesions.
      ,
      • Schwartz G.J.
      Integrative capacity of the caudal brainstem in the control of food intake.
      ]. In contrast, the reward system's primary function is to integrate the motivational and rewarding aspects of eating behavior [
      • Berthoud H.-R.
      • Lenard N.R.
      • Shin A.C.
      Food reward, hyperphagia, and obesity.
      ]; a key brain region is the striatum [
      • Ungerstedt U.
      Adipsia and aphagia after 6-hydroxydopamine induced degeneration of the nigro-striatal dopamine system.
      ]. The homeostatic and reward systems are strongly interconnected, and there are strong anatomical and functional interactions between these regulatory systems [
      • Ong Z.Y.
      • et al.
      Paraventricular thalamic control of food intake and reward: role of glucagon-like peptide-1 receptor signaling.
      ,
      • Ferrario C.R.
      • et al.
      Homeostasis meets motivation in the battle to control food intake.
      ]. The neurotransmitters serotonin and dopamine play important roles in the homeostatic and reward regulation [
      • Leibowitz S.F.
      • Alexander J.T.
      Hypothalamic serotonin in control of eating behavior, meal size, and body weight.
      ,
      • Murray S.
      • et al.
      Hormonal and neural mechanisms of food reward, eating behaviour and obesity.
      ], respectively; however, we note that many other neurotransmitters are involved [
      • Schwartz M.W.
      • et al.
      Central nervous system control of food intake.
      ,
      • Tuominen L.
      • et al.
      Aberrant mesolimbic dopamine-opiate interaction in obesity.
      ]. Serotonin transporters (SERTs) and dopamine transporters (DATs) facilitate the reuptake of serotonin and dopamine from the synaptic cleft and thereby regulate intrasynaptic neurotransmitter levels [
      • Jaber M.
      • et al.
      The dopamine transporter: a crucial component regulating dopamine transmission.
      ,
      • Best J.
      • Nijhout H.F.
      • Reed M.
      Serotonin synthesis, release and reuptake in terminals: a mathematical model.
      ].
      Changes in serotonergic and/or dopaminergic signaling are hypothesized to contribute to obesity [
      • Lam D.D.
      • et al.
      Brain serotonin system in the coordination of food intake and body weight.
      ,
      • Volkow N.D.
      • Wang G.J.
      • Baler R.D.
      Reward, dopamine and the control of food intake: implications for obesity.
      ]. Reduced postprandial serotonergic signaling results in insufficient inhibition of additional food intake (i.e., beyond homeostatic needs) [
      • Jean A.
      • et al.
      Anorexia induced by activation of serotonin 5-HT4 receptors is mediated by increases in CART in the nucleus accumbens.
      ,
      • van Galen K.A.
      • Ter Horst K.W.
      • Serlie M.J.
      Serotonin, food intake, and obesity.
      ]. In turn, postprandial striatal dopamine release is blunted in diet-induced obese mice, suggesting a reduction in the rewarding aspects of food intake; this may promote a compensational increase in food intake [
      • Tellez L.A.
      • et al.
      A gut lipid messenger links excess dietary fat to dopamine deficiency.
      ]. Although methods to directly measure cerebral serotonin and dopamine concentrations in humans in vivo are unavailable, molecular neuroimaging techniques do provide surrogate markers for the assessment of these neurochemicals in humans. For instance, we have previously observed that SERT availability in the thalamus, a region that connects the homeostatic and rewards systems, is dependent on the timing of food intake during a hypocaloric diet intervention [
      • Versteeg R.I.
      • et al.
      Timing of caloric intake during weight loss differentially affects striatal dopamine transporter and thalamic serotonin transporter binding.
      ]. Other studies that have used molecular neuroimaging to measure cerebral serotonergic and dopaminergic signaling also support a role for serotonin and dopamine signaling in human obesity [
      • van Galen K.A.
      • et al.
      The role of central dopamine and serotonin in human obesity: lessons learned from molecular neuroimaging studies.
      ]. Taken together, the data suggest that obesity is associated with decreased tonic serotonin levels, with increased tonic dopamine levels, and with reduced phasic dopamine release in the synapse [
      • van Galen K.A.
      • et al.
      The role of central dopamine and serotonin in human obesity: lessons learned from molecular neuroimaging studies.
      ]. However, these studies were conducted in subjects who had differential basal metabolic states at the time of imaging. This is important, because the results of animal studies suggested effects of fasting duration on the serotonergic and dopaminergic neurotransmitter systems [
      • Roseberry A.G.
      Acute fasting increases somatodendritic dopamine release in the ventral tegmental area.
      ,
      • Patterson T.A.
      • et al.
      Food deprivation decreases mRNA and activity of the rat dopamine transporter.
      ,
      • Brunetti L.
      • et al.
      Effects of ghrelin and amylin on dopamine, norepinephrine and serotonin release in the hypothalamus.
      ]; differences in fasting duration may thus have affected the outcomes of previous human studies.
      From an evolutionary perspective, the metabolic and behavioral responses to foods are essential to survive periods of food scarcity [
      • Kerndt P.R.
      • et al.
      Fasting: the history, pathophysiology and complications.
      ]. Among the responses, the metabolic adaptation to fasting requires a transition from glucose to lipid as the primary energy substrate. In line, the response to fasting is characterized by decreased plasma insulin, glucose, and leptin levels and by increased plasma free fatty acid (FFA), glucagon, ketone body, and ghrelin levels [
      • Kerndt P.R.
      • et al.
      Fasting: the history, pathophysiology and complications.
      ,
      • Boden G.
      • et al.
      Effect of fasting on serum leptin in normal human subjects.
      ,
      • Zhao T.J.
      • et al.
      Ghrelin secretion stimulated by {beta}1-adrenergic receptors in cultured ghrelinoma cells and in fasted mice.
      ]. Data from animal studies suggest that changes in these peripheral hormone and nutrient levels affect the central serotonergic and dopaminergic systems [
      • Brunetti L.
      • et al.
      Effects of ghrelin and amylin on dopamine, norepinephrine and serotonin release in the hypothalamus.
      ,
      • Fernstrom J.D.
      • Wurtman R.J.
      Brain serotonin content: increase following ingestion of carbohydrate diet.
      ,
      • Stouffer M.A.
      • et al.
      Insulin enhances striatal dopamine release by activating cholinergic interneurons and thereby signals reward.
      ,
      • Blum K.
      • Thanos P.K.
      • Gold M.S.
      Dopamine and glucose, obesity, and reward deficiency syndrome.
      ,
      • Khanh D.V.
      • et al.
      Leptin and insulin signaling in dopaminergic neurons: relationship between energy balance and reward system.
      ,
      • Abizaid A.
      Ghrelin and dopamine: new insights on the peripheral regulation of appetite.
      ], thus signaling to the brain when systemic nutrient availability is low. In this regard, obesity has been associated with metabolic inflexibility (i.e., a diminished capacity for mitochondrial fuel switching and an impaired regulation of mitochondrial fuel balance [
      • Goodpaster B.H.
      • Sparks L.M.
      Metabolic flexibility in health and disease.
      ]), a condition that may become especially apparent during prolonged fasting. In the context of metabolic inflexibility, changes in peripheral hormone and nutrient levels may thus affect how the homeostatic circuitry of an individual with obesity adapts to prolonged fasting and/or other nutritional states. Finally, the homeostatic system directly interacts with the reward system to enhance the motivational and rewarding aspects of food intake under fasting conditions, thereby contributing to an integrated behavioral response that increases the likelihood of short-term restoration of energy balance [
      • Berthoud H.R.
      • Munzberg H.
      • Morrison C.D.
      Blaming the brain for obesity: integration of hedonic and homeostatic mechanisms.
      ,
      • Morton G.J.
      • Meek T.H.
      • Schwartz M.W.
      Neurobiology of food intake in health and disease.
      ]. It thus seems that fasting affects both the homeostatic and reward systems, and studying the metabolic and central adaptations to fasting in humans with normal weight or obesity will likely provide novel insights into the central serotonergic and dopaminergic systems, the regulation of eating behavior, and the pathogenesis of obesity.
      Here, we aimed to: i) assess the effects of fasting on central serotonin and dopamine transporter availability, ii) determine differences in the effects of fasting between lean humans vs humans with obesity, and iii) explore the relationships between fasting-induced changes in peripheral metabolic signals, energy substrate oxidation and the central serotonergic and dopaminergic systems.

      2. Methods

      2.1 Design

      This randomized controlled crossover study was designed to study the effects of 12 vs 24 h of fasting on hypothalamic and thalamic SERT availability, striatal DAT availability, and parameters of systemic metabolism in lean subjects and subjects with obesity. The protocol was approved by the Academic Medical Center medical ethics committee. All subjects provided written informed consent in accordance with the Declaration of Helsinki. The study was prospectively registered in the Netherlands Trial Registry (http://www.trialregister.nl: NTR6609).

      2.2 Subjects

      We recruited 10 lean subjects and 10 subjects with obesity from the general population in the Amsterdam metropolitan area via local advertisements. Subjects were eligible to participate if they: i) were men, ii) were aged 50–75 years (to prevent radiation exposure in younger subjects [
      • Sminia P.
      • Lammertsma A.A.
      • Greuter M.
      • Wiegman M.J.
      • de Lange M.
      • de Fluiter-Zeeman M.
      • et al.
      Human exposure to ionising radiation for clinical and research purposes: radiation dose & risk estimates: report 26 van de Nederlandse Commissie voor Stralings Dosimetrie.
      ]), iii) had either a body mass index (BMI) <25 kg/m2 (lean subjects) or BMI >30 kg/m2 (subjects with obesity), and iv) had stable weight (<5% weight change) for at least 3 months prior to the study assessments. Exclusion criteria were: i) use of any medication (except for thyroid hormone, antihypertensive, and/or lipid-lowering drugs); ii) any somatic disorder (except for adequately treated hypothyroidism, hypertension, and/or dyslipidemia); iii) history of any psychiatric or eating disorder; iv) shift work; v) irregular sleeping habits; vi) regular vigorous exercise (>3 h/week); vii) restrained eaters [
      • Herman C.P.
      • Mack D.
      Restrained and unrestrained eating.
      ]; viii) substance abuse (smoking, alcohol >3 units/day, and/or recreational drugs); or ix) any contra-indication for magnetic resonance imaging (MRI). All subjects completed a medical evaluation, including history, physical examination, and blood tests. Systemic insulin sensitivity was estimated using the fasting plasma insulin concentration, where fasting insulin >74 pmol/L indicates insulin resistance [
      • ter Horst K.W.
      • et al.
      Insulin resistance in obesity can be reliably identified from fasting plasma insulin.
      ].

      2.3 Randomization and fasting protocols

      We used a randomized controlled crossover design to compare the effects of 12 vs 24 h of fasting. All subjects underwent two study days: one after a 12-h overnight fast, and one after a 24-h fast that started at 10–12 AM on the day before neuroimaging. Subjects received a sealed envelope, containing instructions to fast for 12 h before the first study day and 24 h before the second study day, or vice versa. Allocation to treatment order was randomized and stratified (1:1) by treatment and by obesity using the GraphPad QuickCalcs website (http://graphpad.com/quickcalcs/randomize2/). The primary investigator was blinded to allocation.
      We defined fasting as abstinence from all foods and drinks, except for tap water. Subjects started their fast exactly 12 or 24 h before the first brain scan (Fig. 1). To limit confounding due to different dietary habits, subjects were instructed to maintain an eucaloric diet for 72 h before starting their fast. The eucaloric diet was identical before both fasting interventions. It was created individually on the basis of resting energy expenditure (REE), as measured by indirect calorimetry (Vmax Encore 29, Carefusion, San Diego, CA, USA) and estimated physical activity. The diet consisted of three meals and two snacks per day, and had a standardized macronutrient ratio of 50% carbohydrates, 30% fat, and 20% proteins.

      2.4 Brain single-photon emission computed tomography (SPECT) imaging

      Hypothalamic and thalamic SERT and striatal DAT availability were assessed using SPECT imaging and the radiotracer 123I-N-ω-fluoropropyl-2β-carboxymethoxy-3β-(4-iodophenyl)nortropane (123I-FP-CIT), a method that we and others have previously validated [
      • Borgers A.J.
      • et al.
      Imaging of serotonin transporters with [123I]FP-CIT SPECT in the human hypothalamus.
      ,
      • Booij J.
      • et al.
      [123I]FP-CIT binds to the dopamine transporter as assessed by biodistribution studies in rats and SPECT studies in MPTP-lesioned monkeys.
      ,
      • Booij J.
      • et al.
      Quantification of striatal dopamine transporters with 123I-FP-CIT SPECT is influenced by the selective serotonin reuptake inhibitor paroxetine: a double-blind, placebo-controlled, crossover study in healthy control subjects.
      ,
      • Booij J.
      • et al.
      One-day protocol for imaging of the nigrostriatal dopaminergic pathway in Parkinson’s disease by [123I]FPCIT SPECT.
      ,
      • Ziebell M.
      • et al.
      Serotonin transporters in dopamine transporter imaging: a head-to-head comparison of dopamine transporter SPECT radioligands 123I-FP-CIT and 123I-PE2I.
      ]. SERTs and DATs facilitate the synaptic reuptake of serotonin and dopamine, respectively, into the pre-synaptic neuron and thereby regulate the amounts of neurotransmitter available for signaling [
      • Torres G.E.
      • Gainetdinov R.R.
      • Caron M.G.
      Plasma membrane monoamine transporters: structure, regulation and function.
      ]. In vivo-assessed SERT and DAT availability reflect a combination of the expression of the transporter (Bmax) and the affinity of the radiotracer for the given transporter (1/Kd [
      • Innis R.B.
      • et al.
      Consensus nomenclature for in vivo imaging of reversibly binding radioligands.
      ]). In other words, an increase in Bmax and a decrease in Kd can both increase 123I-FP-CIT binding (Bmax/Kd). The Kd can thus be influenced by changing the synaptic levels of neurotransmitters or a change in the conformational state of the transporter [
      • Cheng M.H.
      • Bahar I.
      Monoamine transporters: structure, intrinsic dynamics and allosteric regulation.
      ]. An increase in transporter availability (i.e., higher binding of 123I-FP-CIT) reflects higher transporter expression (i.e., higher Bmax) or higher affinity (i.e., lower Kd) induced by a decrease in the concentration of endogenous neurotransmitter, a conformational change, or a combination of these factors. Because 123I-FP-CIT binding in the hypothalamus/thalamus and the striatum can be blocked in vivo by selective SERT and DAT reuptake inhibitors, respectively, binding of 123I-FP-CIT in the hypothalamus/thalamus and the striatum predominantly reflect SERT and DAT, respectively [
      • Borgers A.J.
      • et al.
      Imaging of serotonin transporters with [123I]FP-CIT SPECT in the human hypothalamus.
      ,
      • Booij J.
      • et al.
      [123I]FP-CIT binds to the dopamine transporter as assessed by biodistribution studies in rats and SPECT studies in MPTP-lesioned monkeys.
      ]. We have previously demonstrated that hypothalamic/thalamic SERT and striatal DAT availability can be optimally imaged 2 and 3 h after the administration of the radiotracer, respectively [
      • Booij J.
      • et al.
      One-day protocol for imaging of the nigrostriatal dopaminergic pathway in Parkinson’s disease by [123I]FPCIT SPECT.
      ,
      • Koopman K.E.
      • et al.
      Assessing the optimal time point for the measurement of extrastriatal serotonin transporter binding with 123I-FP-CIT SPECT in healthy, male subjects.
      ].
      Subjects were administered an intravenous bolus of approximately 100 MBq 123I-FP-CIT (specific activity >750 MBq/nmol; radiochemical purity >98%; produced in accordance with GMP guidelines; GE Healthcare, Eindhoven, The Netherlands). Next, SPECT imaging was performed using the InSPira HD system, a brain-dedicated SPECT camera (Neurologica, Boston, USA) with the following parameters: acquisition time per slice, 180 s; slice thickness, 4 mm. Slices were acquired from the level of the cerebellum up to the striatum. SPECT images were reconstructed with an iterative expectation maximization algorithm and corrected for attenuation by manually aligning an adult head template [
      • Booij J.
      • et al.
      [123I]FP-CIT binds to the dopamine transporter as assessed by biodistribution studies in rats and SPECT studies in MPTP-lesioned monkeys.
      ,
      • Boot E.
      • et al.
      AMPT-induced monoamine depletion in humans: evaluation of two alternative [123I]IBZM SPECT procedures.
      ]. To limit thyroid uptake of free radioactive iodide, all subjects were pre-treated with potassium iodide.

      2.5 Brain MRI scanning

      For anatomical reference, a T1-weighted (T1w) MRI scan of the brain was obtained on a 3.0-T Ingenia MR scanner (Philips Healthcare, Best, The Netherlands) using a 32-channel receive-only head coil and the following scan parameters: TR/TE 7/3.18 ms; flip angle 9°; 1 mm isotropic resolution. Individual SPECT scans were co-registered to individual MRI scans using 6-parameter rigid body registration in SPM12 (Wellcome Centre for Neuroimaging, London, UK) (Fig. 2). To optimize registration, a high-intensity striatal mask was superimposed on the T1w scan to resemble the contrast on the SPECT scan.
      Fig. 2
      Fig. 2Representative T1w anatomical brain MRI scans overlaid with co-registered SPECT images and ROI masks. Striatal (red mask), hypothalamic (blue mask), and thalamic (green mask) uptake of the radiotracer 123I-FP-CIT.

      2.6 Region-of-interest (ROI) analysis

      Hypothalamic and thalamic SERT as well as striatal DAT availability were quantified using ROI analysis. We used Freesurfer version 5.3.0 [
      • Dale A.M.
      • Fischl B.
      • Sereno M.I.
      Cortical surface-based analysis. I. Segmentation and surface reconstruction.
      ,
      • Fischl B.
      • et al.
      Whole brain segmentation: automated labeling of neuroanatomical structures in the human brain.
      ] to obtain thalamic and striatal masks from individual T1w MRI scans. Hypothalamic masks were outlined manually using anatomical landmarks on ITK-SNAP version 3.4.0 [
      • Borgers A.J.
      • et al.
      Imaging of serotonin transporters with [123I]FP-CIT SPECT in the human hypothalamus.
      ,
      • Yushkevich P.A.
      • et al.
      User-guided 3D active contour segmentation of anatomical structures: significantly improved efficiency and reliability.
      ,
      • Swaab D.F.
      • et al.
      Functional neuroanatomy and neuropathology of the human hypothalamus.
      ]. The cerebellum was used to quantify nonspecific radiotracer activity; 123I-FP-CIT activity in this region does not reflect binding to either SERTs or DATs, since the cerebellum is almost devoid of these transporters [
      • Booij J.
      • et al.
      [123I]FP-CIT binds to the dopamine transporter as assessed by biodistribution studies in rats and SPECT studies in MPTP-lesioned monkeys.
      ,
      • Parsey R.V.
      • et al.
      Metabolite considerations in the in vivo quantification of serotonin transporters using 11C-DASB and PET in humans.
      ,
      • Hall H.
      • et al.
      Visualization of the dopamine transporter in the human brain postmortem with the new selective ligand [125I]PE2I.
      ]. We obtained cerebellar masks by warping the cerebellum (without vermis) from the Harvard-Oxford subcortical atlas (https://fsl.fmrib.ox.ac.uk/fsl/fslwiki/Atlases) to individual T1w MRI using FSL (FMRIB Software Library, version 5.0.8, Oxford, UK). For each ROI, we measured the availability of regional SERT and DAT [i.e., binding potential (BPND) of 123I-FP-CIT] by calculating the specific-to-nonspecific binding ratio, using the following formula: BPND = (mean hypothalamic, thalamic or striatal binding – mean cerebellar binding)/mean cerebellar binding. Fasting-induced changes in SERT and DAT availability were defined as BPND after a 24-h fast, relative to BPND after a 12-h fast.

      2.7 Metabolic effects of fasting

      Venous blood samples were drawn after both fasting interventions to determine the effect of a 12-h vs 24-h fast on circulating hormone and nutrient levels. In addition, indirect calorimetry was performed to measure REE and the respiratory quotient (RQ) after 12 vs 24 h of fasting.

      2.8 Laboratory analysis

      Plasma glucose was determined with the glucose oxidase method using a Biosen C-line plus glucose analyzer (EKF Diagnostics, Barleben/Magdeburg, Germany). Plasma insulin was determined by immunoassay on an Atellica system (Siemens, Erlangen, Germany) with intra-assay variation of 3% and inter-assay variation of 7%. Plasma FFAs were determined by an enzymatic colorimetric method (NEFA C test kit; Wako Chemicals, Neuss, Germany) with intra-assay variation of 1% and inter-assay variation of 4–15%. Plasma glucagon was determined by radioimmunoassay (Linco Research, St Charles, MO, USA) with intra-assay variation of 4–8% and inter-assay variation of 6–11%. Plasma leptin was determined by radioimmunoassay (Millipore, Burlington, MA, USA) with intra-assay and inter-assay variation of 6%. Plasma ghrelin was determined by radioimmunoassay (Millipore, Burlington, MA, USA) with intra-assay variation of 4% and inter-assay variation of 6%.

      2.9 Statistical analysis

      Data are presented as mean ± standard deviation (SD) or median [interquartile range (IQR)], unless stated otherwise. Data were assessed for normality by inspection of histograms; outliers were detected using Grubbs tests (upper 2.5% significance level). Outcomes were evaluated using a repeated-measures analysis of variance (ANOVA), with fasting duration as the within-subjects factor and group (lean men vs men with obesity) as the between-subjects factor. Differences between groups were evaluated using t-tests or Mann-Whitney U tests. Fasting-induced changes within groups were assessed using paired t-tests or Wilcoxon signed-rank tests. Correlations were evaluated using Pearson's or Spearman's coefficient. Multiple linear regression analysis was performed to assess the independent associations between peripheral fasting signals and regional SERT or DAT availability. Findings were considered significant if p < 0.05. SPSS version 26.0 (IBM, Armonk, NY, USA) was used for all statistical testing.

      3. Results

      3.1 Participants

      Ten lean men and nine men with obesity completed the study (Table 1). One individual with obesity was excluded, because his SPECT scans could not be analyzed due to technical issues. Groups did not differ in age. Subjects with obesity had higher fasting plasma insulin concentrations, indicating insulin resistance [
      • ter Horst K.W.
      • et al.
      Insulin resistance in obesity can be reliably identified from fasting plasma insulin.
      ], and higher fasting leptin concentrations. There were no differences between groups in hypothalamic and thalamic SERT and striatal DAT availability after the 12-h fast.
      Table 1Characteristics of study participants after the 12-h fast (n = 19).
      Lean men (n = 10)Men with obesity (n = 9)p
      Age (years)64 ± 765 ± 80.828
      Length (cm)174 ± 9177 ± 60.499
      Weight (kg)70 ± 11102 ± 14<0.001
      BMI (kg/m2)23.0 ± 1.832.6 ± 2.8<0.001
      Waist circumference (cm)90 ± 9117 ± 12<0.001
      REE (kcal/kg/day)20.2 ± 3.818.6 ± 2.50.283
      RQ0.78 ± 0.070.77 ± 0.050.737
      Glucose (mmol/L)4.8 ± 0.45.5 ± 1.40.127
      FFA (mmol/L)0.48 [0.38–0.63]0.45 [0.35–0.58]0.653
      Insulin (pmol/L)28 [26–48]130 [58–170]0.002
      Glucagon (ng/L)87 [70–116]88 [70–133]0.595
      Leptin (μg/L)6 [5–9]23 [16–33]<0.001
      Ghrelin (pg/mL)33.8 [19.8–47.3]13.7 [<6–28.5]0.060
      Striatal DAT BPND4.47 ± 1.314.87 ± 1.220.501
      Hypothalamic SERT BPND0.32 [0.18–0.53]0.45 [0.33–0.71]0.248
      Thalamic SERT BPND0.60 [0.55–0.69]0.53 [0.36–0.62]0.165
      Data are mean ± SD or median [IQR] and were using t-tests or Mann-Whitney U tests.

      3.2 Metabolic effects of fasting

      The 24-h fast had significant effects on parameters of systemic metabolism (Table 2), indicating ongoing metabolic adaptations when fasting duration is extended from 12 to 24 h. As compared with the 12-h fast, the 24-h fast induced reductions in plasma glucose and leptin concentrations in both groups. In lean men, plasma insulin concentrations and the RQ also decreased, with the latter indicating a shift from carbohydrate towards lipid oxidation. In subjects with obesity, plasma insulin concentrations and the RQ did not decrease after 24, as compared with 12, hours of fasting, which is consistent with the hypothesis that obesity is associated with metabolic inflexibility [
      • Astrup A.
      The relevance of increased fat oxidation for body-weight management: metabolic inflexibility in the predisposition to weight gain.
      ]. In neither group, REE nor plasma FFA, glucagon, and ghrelin levels differed between the fasting durations. There were no between-group differences in the effects of prolonged fasting on fasting metabolites or hormones. As compared to the men with obesity, lean men tended to show a stronger effect of prolonged fasting on REE and RQ.
      Table 2The effects of a 24-h fast on metabolic and neuroimaging parameters, as compared with a 12-h fast.
      Lean men (n = 10)Men with obesity (n = 9)p
      For lean men vs men with obesity on t-test or Mann Whitney U test.
      REE (%)9.0 ± 16.5−3.4 ± 7.00.054
      RQ (%)−10.9 [−20.3 to −2.7]
      p < 0.05 for within-group difference on paired t-test or Wilcoxon signed rank test.
      0.0 [−13.3–6.0]0.063
      Fasting glucose (%)−9.7 ± 7.9
      p < 0.05 for within-group difference on paired t-test or Wilcoxon signed rank test.
      −6.7 ± 5.6
      p < 0.05 for within-group difference on paired t-test or Wilcoxon signed rank test.
      0.360
      Fasting FFA (%)80.4 ± 101.226.6 ± 40.40.155
      Fasting insulin (%)−40.3 [−58.6 to −19.2]
      p < 0.05 for within-group difference on paired t-test or Wilcoxon signed rank test.
      −28.6 [−33.2–9.8]0.133
      Fasting glucagon (%)3.3 ± 7.616.3 ± 21.40.088
      Fasting leptin (%)−40.9 ± 18.6
      p < 0.05 for within-group difference on paired t-test or Wilcoxon signed rank test.
      −22.0 ± 21.7
      p < 0.05 for within-group difference on paired t-test or Wilcoxon signed rank test.
      0.056
      Fasting ghrelin (%)−29.2 [−47.3–13.1]0.0 [−3.3–8.3]0.156
      Striatal DAT BPND (%)4.3 [−4.9–21.3]−9.9 [−23.0–20.3]0.327
      Hypothalamic SERT BPND (%)48.3 ± 76.4
      p < 0.05 for within-group difference on paired t-test or Wilcoxon signed rank test.
      −36.3 ± 71.10.029
      Thalamic SERT BPND (%)−8.9 ± 28.1−2.0 ± 44.00.685
      Data are mean ± SD or median [IQR] and expressed as percentage difference between the 12-h and 24-h fasting interventions.
      a p < 0.05 for within-group difference on paired t-test or Wilcoxon signed rank test.
      b For lean men vs men with obesity on t-test or Mann Whitney U test.

      3.3 Effects of fasting on central SERT and DAT availability

      To study the effects of fasting on central SERT and DAT availability, we performed SPECT scans after 12 vs 24 h of fasting. We observed no overall differences in hypothalamic and thalamic SERT or in striatal DAT availability after the 24-h fast, as compared with the 12-h fast (Fig. 3A–C ). However, as can be appreciated from the individual subjects' data (Fig. 3A–C), we did observe high inter-individual variation in the effects of fasting. In this regard, we observed an important interaction between obesity and fasting duration on hypothalamic SERT availability (F1,16 = 6.474, p = 0.022, Fig. 3D, Table 2), indicating that fasting duration differentially affects hypothalamic SERT availability in lean individuals vs individuals with obesity. In lean men, hypothalamic SERT availability significantly increased by 48.3% ± 76.4% (p = 0.044) upon 24 h of fasting, which is in contrast to the unchanged hypothalamic SERT availability (−36.3% ± 71.1%, p = 0.179) in men with obesity (Table 2). We observed no interactions between obesity and fasting on thalamic SERT (F1,17 = 0.265, p = 0.614, Fig. 3E) or striatal DAT availability (F1,17 = 1.989, p = 0.176, Fig. 3F), suggesting that the high inter-individual variation in the effects of fasting on thalamic SERT and striatal DAT availability is not explained by differences in body weight.
      Fig. 3
      Fig. 3Radiotracer binding after a 12-h vs 24-h fast in lean men and men with obesity. (A) Hypothalamic SERT availability. (B) Thalamic SERT availability. (C) Striatal DAT availability. Mean radiotracer binding after a 12-h and 24-h fast in lean men and men with obesity. (D) Hypothalamic SERT availability. (E) Thalamic SERT availability. (F) Striatal DAT availability. Data are individual subjects (A–C) and mean ± SD (D–F). One outlier was removed (A + D). *p < 0.05 for paired t-test. **p < 0.05 for fasting-obesity interaction on repeated measures ANOVA.

      3.4 Predictors of central SERT and DAT availability in fasting humans

      The metabolic adaptation to fasting includes changes in multiple plasma hormone and nutrient concentrations, all of which may signal nutritional depletion to the brain [
      • Brunetti L.
      • et al.
      Effects of ghrelin and amylin on dopamine, norepinephrine and serotonin release in the hypothalamus.
      ,
      • Fernstrom J.D.
      • Wurtman R.J.
      Brain serotonin content: increase following ingestion of carbohydrate diet.
      ,
      • Stouffer M.A.
      • et al.
      Insulin enhances striatal dopamine release by activating cholinergic interneurons and thereby signals reward.
      ,
      • Blum K.
      • Thanos P.K.
      • Gold M.S.
      Dopamine and glucose, obesity, and reward deficiency syndrome.
      ,
      • Khanh D.V.
      • et al.
      Leptin and insulin signaling in dopaminergic neurons: relationship between energy balance and reward system.
      ,
      • Abizaid A.
      Ghrelin and dopamine: new insights on the peripheral regulation of appetite.
      ]. Therefore, we next explored if these changes might contribute to the observed inter-individual variation in transporter availability upon fasting. In all subjects, there were no correlations between fasting-induced changes in transporter availability and circulating glucose, insulin, glucagon, leptin, and ghrelin levels (not shown). Changes in plasma FFA levels correlated positively with changes in hypothalamic SERT (Fig. 4A ) and negatively with changes in striatal DAT availability (Fig. 4B), indicating that a fasting-induced increase in plasma FFA levels was associated with an increase in hypothalamic SERT availability and with a decrease in striatal DAT availability. Plasma FFA levels did not correlate with thalamic SERT availability (r = 0.135, p = 0.580). Because increased plasma FFA levels reflect ongoing lipolysis and a crucial shift from carbohydrate to lipid metabolism during prolonged fasting, these data may indicate a role for circulating FFAs as a signal of systemic carbohydrate depletion to the central serotoninergic and dopaminergic systems.
      Fig. 4
      Fig. 4Fasting-induced changes in circulating FFAs predict central SERT and DAT availability upon prolonged fasting in humans. Scatterplots showing the relationship between fasting-induced changes in plasma FFA levels and (A) hypothalamic SERT availability or (B) striatal DAT availability in all subjects. Data are lean (●) subjects or subjects with obesity (○) (A–B). One outlier was removed (A).

      3.5 Relation with insulin resistance

      Individuals with peripheral insulin resistance also show blunted cerebral responses to insulin, suggesting central insulin resistance [
      • Anthony K.
      • et al.
      Attenuation of insulin-evoked responses in brain networks controlling appetite and reward in insulin resistance: the cerebral basis for impaired control of food intake in metabolic syndrome?.
      ,
      • Kullmann S.
      • et al.
      Brain insulin sensitivity is linked to adiposity and body fat distribution.
      ]. We hypothesized that normal insulin sensitivity is required to sense fasting-induced decreases in plasma insulin levels. To this end, we divided all subjects into an insulin-sensitive and an insulin-resistant group (Supplemental Table S1), using fasting plasma insulin concentrations as described [
      • ter Horst K.W.
      • et al.
      Insulin resistance in obesity can be reliably identified from fasting plasma insulin.
      ]. In the 13 subjects with insulin sensitivity, changes in plasma insulin levels tended to negatively correlate with changes in striatal DAT availability (rho = −0.527, p = 0.064), suggesting that a decrease in plasma insulin levels was associated with an increase in striatal DAT availability. This was not observed in the six subjects with insulin resistance (rho = 0.143, p = 0.787). Changes in plasma insulin levels did not correlate with hypothalamic and thalamic SERT availability in the insulin-sensitive (rho = −0.154, p = 0.633 and rho = 0.071, p = 0.817, respectively) or insulin-resistant subjects (rho = −0.714, p = 0.111 and rho = −0.657, p = 0.156, respectively).
      Insulin inhibits cerebral FFA oxidation [
      • Lam T.K.
      • Schwartz G.J.
      • Rossetti L.
      Hypothalamic sensing of fatty acids.
      ]; fasting-induced reductions in insulin signaling may thus increase neuronal FFA oxidation. We therefore hypothesized that changes in FFA and insulin levels together improve the accuracy of the prediction of changes in striatal DAT availability, at least in the subjects with insulin sensitivity. Indeed, multiple regression analysis showed that changes in plasma FFAs and insulin both significantly predicted changes in striatal DAT availability in insulin-sensitive subjects (Fig. 5A–B ), together accounting for 44% of the observed variation (Supplemental Table S2). This finding is consistent with the concept that both low plasma insulin and high plasma FFAs may directly or indirectly provide a fasting signal to the central reward system.
      Fig. 5
      Fig. 5Fasting-induced changes in plasma FFA and insulin together accounted for 44% of the variation in striatal DAT availability (adj. R2 = 0.441, p = 0.022). Partial regression plots showing the relationship between fasting-induced changes in striatal DAT availability and (A) plasma FFA levels, after adjusting for plasma insulin levels or (B) plasma insulin levels, after adjusting for plasma FFA levels. Data represent the subset of insulin-sensitive subjects (n = 13).

      4. Discussion

      To the best of our knowledge, this is the first study to assess the effects of fasting on the central serotoninergic and dopaminergic systems in humans. Data from our study demonstrate that: i) a 24-h fast increases hypothalamic SERT availability in lean men, but not in men with obesity; ii) 24 h of fasting does not significantly affect mean thalamic SERT or striatal DAT availability in lean men nor in men with obesity, but with high inter-individual variation in the observed responses; and iii) fasting-induced changes in plasma FFA and insulin levels account for much of the observed variation.
      Fasting differentially affected hypothalamic SERT availability in lean subjects vs subjects with obesity: in lean subjects, hypothalamic SERT availability increased upon the 24-h fast, whereas in individuals with obesity, hypothalamic SERT availability did not change significantly. An increase in hypothalamic SERT availability upon 24 h of fasting likely points to a decrease in hypothalamic serotonergic signaling: increased radiotracer binding either reflects increased synaptic SERT expression, which promotes reuptake of serotonin from the synapse, and/or it reflects increased radiotracer affinity for SERT induced by decreased synaptic serotonin levels [
      • Innis R.B.
      • et al.
      Consensus nomenclature for in vivo imaging of reversibly binding radioligands.
      ,
      • Grouleff J.
      • et al.
      Monoamine transporters: insights from molecular dynamics simulations.
      ]. In rodents and healthy humans, a short-term increase in serotonergic signaling reduces food intake, suggesting a role in satiety [
      • Lam D.D.
      • Heisler L.K.
      Serotonin and energy balance: molecular mechanisms and implications for type 2 diabetes.
      ,
      • Voigt J.P.
      • Fink H.
      Serotonin controlling feeding and satiety.
      ]. In contrast, a decrease in serotonergic signaling stimulates food-seeking behavior and intake in rodents [
      • Waldbillig R.J.
      • Bartness T.J.
      • Stanley B.G.
      Increased food intake, body weight, and adiposity in rats after regional neurochemical depletion of serotonin.
      ,
      • Saller C.F.
      • Stricker E.M.
      Hyperphagia and increased growth in rats after intraventricular injection of 5,7-dihydroxytryptamine.
      ,
      • Breisch S.T.
      • Zemlan F.P.
      • Hoebel B.G.
      Hyperphagia and obesity following serotonin depletion by intraventricular p-chlorophenylalanine.
      ]. Given the anorexigenic effects of hypothalamic serotonergic signaling, the fasting-induced increase in hypothalamic SERT availability in the lean healthy subjects likely reflects decreased serotonergic signaling, which is consistent with an adequate response to prolonged fasting that would contribute to the promotion of food-seeking behavior and restoration of energy balance [
      • Lam D.D.
      • et al.
      Brain serotonin system in the coordination of food intake and body weight.
      ]. In individuals with obesity, however, prolonged fasting did not change hypothalamic SERT availability. This may be explained by chronic perturbation in serotonergic signaling in obesity per se or by a difference in the overall metabolic response to 24 h of fasting [
      • van Galen K.A.
      • et al.
      The role of central dopamine and serotonin in human obesity: lessons learned from molecular neuroimaging studies.
      ,
      • Strombom U.
      • et al.
      The concentrations of monoamine metabolites and neuropeptides in the cerebrospinal fluid of obese women with different body fat distribution.
      ].
      We found that plasma FFA levels were the strongest predictors of hypothalamic SERT and striatal DAT availability. Circulating FFA levels increase during fasting due to the stimulation of adipose tissue lipolysis, and FFAs serve as an alternative fuel source [
      • Gordon Jr., R.S.
      • Cherkes A.
      Unesterified fatty acid in human blood plasma.
      ]. Our data suggest that circulating FFAs may also function as a signal of nutritional depletion for the brain. In this regard, FFAs can enter the brain from the peripheral circulation, with plasma and brain concentrations of some FFAs showing direct correlations [
      • Mitchell R.W.
      • Hatch G.M.
      Fatty acid transport into the brain: of fatty acid fables and lipid tails.
      ,
      • Freund Levi Y.
      • et al.
      Transfer of omega-3 fatty acids across the blood-brain barrier after dietary supplementation with a docosahexaenoic acid-rich omega-3 fatty acid preparation in patients with Alzheimer’s disease: the OmegAD study.
      ]. FFAs are natural ligands of free fatty acid receptors (FFARs), a group of orphan G-protein-coupled receptors (GPCRs) involved in the regulation of energy homeostasis [
      • Kimura I.
      • et al.
      Free fatty acid receptors in health and disease.
      ]. Now, we demonstrate that a fasting-induced increase in plasma FFA levels is associated with an increase in hypothalamic SERT availability. Therefore, our data are consistent with the interpretation that FFAs are a direct signal of nutritional scarcity to the brain, contributing to behavioral adaptation through the central serotonergic system.
      A fasting-induced increase in plasma FFA levels is also associated with a decrease in striatal DAT availability. In rodents, fasting decreases striatal DAT function [
      • Patterson T.A.
      • et al.
      Food deprivation decreases mRNA and activity of the rat dopamine transporter.
      ,
      • Zhen J.
      • Reith M.E.
      • Carr K.D.
      Chronic food restriction and dopamine transporter function in rat striatum.
      ], reducing the re-uptake of dopamine from the synaptic cleft. In line with this, DAT knock-out mice show increased motivation to obtain food [
      • Pecina S.
      • et al.
      Hyperdopaminergic mutant mice have higher “wanting” but not “liking” for sweet rewards.
      ]. In humans, reduced striatal DAT availability is associated with a faster response to a visual food stimulus, indicating enhanced attention for food [
      • Koopman K.E.
      • et al.
      Brain dopamine and serotonin transporter binding are associated with visual attention bias for food in lean men.
      ]. Our present observations may suggest that a reduction in striatal DATs enhances the awareness for food-related stimuli, which, in turn, contributes to the increased likelihood of short-term food consumption. However, a role for hypothalamic/striatal FFA signaling in fasting-induced increases in the motivation for food may seem contrary to studies reporting an anorexigenic effect of intracerebroventricular infusions of select FFAs [
      • Morgan K.
      • Obici S.
      • Rossetti L.
      Hypothalamic responses to long-chain fatty acids are nutritionally regulated.
      ,
      • Obici S.
      • et al.
      Central administration of oleic acid inhibits glucose production and food intake.
      ]. In rodents, insulin and glucose inhibit hypothalamic β-oxidation of FFAs, resulting in the cellular accumulation of FFAs and decreased food intake [
      • Lam T.K.
      • Schwartz G.J.
      • Rossetti L.
      Hypothalamic sensing of fatty acids.
      ,
      • Obici S.
      • et al.
      Inhibition of hypothalamic carnitine palmitoyltransferase-1 decreases food intake and glucose production.
      ]. In the fasting state, however, systemic glucose and insulin levels are low, and the accumulation of FFAs is prevented by increased β-oxidation; this interaction may signal nutritional deprivation to the brain [
      • Lam T.K.
      • Schwartz G.J.
      • Rossetti L.
      Hypothalamic sensing of fatty acids.
      ]. Taken together, we show that fasting-induced increases in plasma FFA levels are associated with changes in both the central serotonergic and dopaminergic systems that may increase the likelihood of short-term food intake.
      Alongside changes in plasma FFA levels, changes in circulating insulin levels contributed to the prediction of fasting-induced changes of striatal DAT availability in insulin-sensitive subjects. In rodents, a growing body of literature supports a role for insulin signaling to the mesolimbic dopamine system in the adaption of motivational and rewarding aspects of food intake to homeostatic needs [
      • Liu S.
      • Borgland S.L.
      Insulin actions in the mesolimbic dopamine system.
      ]. Interestingly, high-fat diet-induced insulin resistance abolished the effects of insulin on striatal dopamine release and reuptake, which could be restored with an insulin receptor sensitizing agent [
      • Fordahl S.C.
      • Jones S.R.
      High-fat-diet-induced deficits in dopamine terminal function are reversed by restoring insulin signaling.
      ]. In humans, insulin receptor expression is high in mesolimbic brain regions [
      • Adem A.
      • et al.
      Insulin-like growth factor 1 (IGF-1) receptors in the human brain: quantitative autoradiographic localization.
      ]. Humans with systemic insulin resistance have been reported to show attenuated insulin-evoked brain responses [
      • Anthony K.
      • et al.
      Attenuation of insulin-evoked responses in brain networks controlling appetite and reward in insulin resistance: the cerebral basis for impaired control of food intake in metabolic syndrome?.
      ]. Intact insulin sensitivity may be required to sense fasting-induced decreases in circulating insulin levels. Thus, our findings suggest that insulin may play a role in the adaptation of the dopaminergic system to fasting in insulin-sensitive humans.
      Previous studies that have assessed the cerebral serotonergic and dopaminergic systems in humans have done so following different durations of fasting [
      • van Galen K.A.
      • et al.
      The role of central dopamine and serotonin in human obesity: lessons learned from molecular neuroimaging studies.
      ]. This complicates the interpretation of those studies, because it is unknown how fasting may have affected the systems under investigation. Here, we show that, the effects of 12 vs 24 h of fasting on hypothalamic SERT availability differ between lean men and men with obesity. In addition, although mean thalamic and striatal transporter availability did not differ after 12 vs 24 h of fasting, we did observe high inter-individual variation in transporter availability after 12 vs 24 h of fasting. In this regard, individual variation in fasting FFA and insulin levels accounted for much of the observed variation. However, given that parts of the variation in the effects of fasting remain unexplained, it seems fair to assume that unmeasured parameters of fasting influence the serotonergic and dopaminergic systems as well. For instance, an increase in ketone bodies may also signal ‘fasting’ to the brain [
      • Owen O.E.
      • et al.
      Brain metabolism during fasting.
      ]. We therefore recommend that investigators consider possible effects of fasting on the serotonergic and dopaminergic systems and standardize fasting duration prior to SPECT imaging in future studies.
      This study was designed to study the effects of prolonged fasting on central SERT and DAT availability, and we acknowledge that our findings on the predictors of central SERT and DAT availability should be interpreted as secondary endpoints. We also note that some of our observations may have been influenced by unmeasured signals of fasting, including ketone bodies. In addition, we only studied men and our findings cannot be extrapolated to women or people in different age groups. Finally, most of the subjects with obesity were classified as class I obesity [
      • Organization, W.H
      Factsheet: obesity and overweight.
      ] and we cannot draw conclusion on individuals with more severe obesity. Going forward, it will be of interest to directly assess how FFAs and insulin signal to the hypothalamic serotoninergic and striatal dopaminergic systems, for instance during infusion or hyperinsulinemic clamp experiments. Finally, due to the limited spatial resolution of the dedicated brain tomographic SPECT system, it was not possible to examine subregional differences within the hypothalamus.
      In conclusion, we show that prolonged fasting increases hypothalamic SERT availability in men with normal weight, but not in men with obesity. We observed no overall effects of fasting on thalamic SERT or striatal DAT availability, but fasting-induced responses in these regions showed high inter-individual variation. Fasting-induced increases in plasma FFAs were associated with increased hypothalamic SERT and decreased striatal DAT availability, and changes in circulating FFA and insulin concentrations together accounted for almost half of the dopaminergic response to fasting. Taken together, the data indicate that human obesity is associated with a blunted hypothalamic serotonergic response to fasting and that FFAs and insulin might serve as important peripheral messengers to the brain to relay information on the nutritional state.

      Funding

      No specific funding was received for the present study.

      CRediT authorship contribution statement

      K.A.G., J.B., K.W.H., and M.J.S. designed the research; K.A.G. performed the research and drafted the manuscript; K.A.G., A.S., S.M.A., and U.A.U. analyzed the data; A.S., J.B., S.E.F., K.W.H., G.J.S., R.J.L. and M.J.S. provided important intellectual input; K.W.H. and M.J.S. had primary responsibility for the final content; and all authors read, revised, and approval of the final manuscript.

      Declaration of competing interest

      The authors declare that there is no conflict of interest associated with the manuscript.

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