A decrease found in both phasic dopamine release and insulin sensitivity after timed-bromocriptine administration via Parlodel in Type 2 Diabetes Mellitus patients (single-patient study)

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A decrease found in both phasic dopamine release and insulin

sensitivity after timed-bromocriptine administration via Parlodel

in Type 2 Diabetes Mellitus patients (single-patient study)

—————————————————————

Anne M.F. Dijker

Written for the journal: Diabetes Care

Name student: Anne Maria Francisca Dijker

Student ID: 11288604

Name supervisors: Mireille Serlie and Jamie van Son

Hand-in date: 21-05-2020

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Index

- Abstract ………... 3

- Introduction ………... 4

- Material and methods ……… 7

- Results ………... 11

- Discussion ……….14

- Acknowledgements ……….…. 18

- References ……… 19

- Appendix 1. Inclusion and exclusion criteria ………... 23

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Abstract

OBJECTIVE - Bromocriptine is a pharmacological option for treating type 2 diabetes mellitus

(T2DM) by simulating the dopamine morning peak. Reinstatement of this peak is believed to increase phasic dopamine release. However, the exact effect of this drug on the circadian dopaminergic rhythm is still unknown. Furthermore, it is assumed that an association between phasic dopamine release and insulin sensitivity is present. Therefore, this study aimed to assess the effect of bromocriptine on phasic dopamine release (measured as dexamphetamine-induced

dopamine release), and whether possible improvement of this release is associated with an increase in insulin sensitivity (measured as exogenous glucose infusion rate).

RESEARCH DESIGN AND METHODS - The dopamine morning peak was simulated in 1 male

Caucasian subject with T2DM via a 12-week timed-bromocriptine administration.

Dexamphetamine-induced dopamine release was assessed using [123_{I]IBZM SPECT imaging and}

exogenous glucose infusion rate using a two-step hyperinsulinemic, euglycemic clamp. In addition, feeding behavior was determined via neuropsychological questionnaires. To determine possible changes in parameters, assessments were repeated after the intervention.

RESULTS - Qualitative analysis showed a decrease in dexamphetamine-induced dopamine release

as well as in exogenous infusion rate of glucose after the intervention in comparison to baseline. In addition, no changes in feeding behavior were seen after the bromocriptine intervention.

CONCLUSIONS - Phasic dopamine release and insulin sensitivity both reduced after 12 weeks of

timed-bromocriptine administration. However, considering the contrasting results in comparison to previous studies, further trials are needed to validate these effects of bromocriptine. Gaining insight in these effects of bromocriptine could contribute to a novel treatment for T2DM.

KEYWORDS: type 2 diabetes mellitus, bromocriptine, phasic dopamine release, insulin

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1. Introduction

Type II Diabetes Mellitus (T2DM) and obesity have become increasingly prevalent over the years (1). This is partly due to the ‘western diet’, rich in fat and sugars, which contributes to the increase in obesity (2). Since obesity is a major risk factor for T2DM, the prevalence of T2DM also

increased over the years (3,4). With an estimated 463 million people diagnosed with T2DM worldwide in 2019, 700 million people are expected to be diagnosed by 2045 (5).

T2DM is characterized by beta-cell failure, and insulin resistance leading to hyperglycemia. Insulin is mainly responsible for the maintenance of normal blood glucose levels enabled by stimulation of glucose uptake by the adipose tissue, liver and skeletal muscles (6). Due to insulin resistance, the adipose tissue, liver and skeletal muscles become less sensitive to insulin. Consequently, the body does not respond to the high insulin levels which results in less glucose uptake and elevated blood glucose levels.

Additional research has shown that, besides the increased insulin levels, dopamine levels of the peri-suprachiasmatic nucleus (peri-SCN) and suprachiasmatic nucleus (SCN) are diminished in obese, insulin-resistant rats in comparison to thin, insulin-sensitive rats (7). The SCN is the central biological clock that regulates oscillations in multiple vital functions of the body, of which

neurohormone secretion is one (8). These oscillations follow a 24-hour rhythm; the circadian rhythm. Dopamine and insulin secretion normally follow such a rhythm. The circadian rhythm of dopamine secretion starts with an increase in dopamine release in the morning, causing a dopamine morning peak. This peak results in a decrease in tonic dopamine levels and enhances phasic

responsiveness to certain stimuli, such as insulin (9). This increased phasic dopamine response is assumed to be needed for improved insulin sensitivity shortly after awakening (10). Thus, when the dopamine morning peak is diminished, the phasic response is decreased which is thought to effect insulin sensitivity. This has been shown in a study where diminished circadian dopamine release to

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the peri-SCN led to fattened, insulin-resistant and glucose-intolerant rats (10). Another study found that lesions of dopamine neurons in the peri-SCN and SCN containing D2 receptors show to induce

insulin resistance in Syrian hamsters (11). Additionally, D2 receptor knockout male mice showed

deterioration of insulin response to glucose (12). These D2 receptors show to have the highest

density in the striatum (13). These results suggest that the dopaminergic input to the peri-SCN and SCN and activation of striatal D2 receptors are necessary for insulin sensitivity. In addition, this

indicates that an association between dopamine release and insulin sensitivity is present. Not only has this association been found in animals, also humans show this phenomenon. Healthy individuals showed impairment in insulin sensitivity as a result of less endogenous dopamine at the D2/3

receptor in the ventral striatum (14).

Although dopamine release and insulin sensitivity show to be associated, it is unknown what exact effect simulation of the dopamine morning peak will have on the phasic dopaminergic

responsiveness and if improvement of this responsiveness is associated with an increase in insulin sensitivity in T2DM patients. Increasing the dopamine morning peak, and therefore attempting to restore the circadian rhythm of dopamine, can be established by early morning administration of bromocriptine, a dopamine D2 receptor agonist (10). This agonist is already used as a monotherapy

for Parkinson’s disease (15) and the effect on certain aspects of T2DM have been studied. Firstly, bromocriptine shows to decrease postprandial glucose levels in T2DM patients without increasing the plasma insulin levels (10). This implicates an improvement of glycemic control. Secondly, a quick-release administration of bromocriptine (bromocriptine-QR therapy), that is ingested within 2 hours of awakening in the morning, results in improved glucose tolerance in T2DM patients (16). This has also been shown in multiple animal models of T2DM (10). Bromocriptine-QR therapy in T2DM patients furthermore reveals a reduction of glycated hemoglobin (HbA1C) which is used as a marker for insulin resistance (16,17). Therefore, it can be expected that bromocriptine improves insulin sensitivity. Thus, bromocriptine demonstrates to modulate certain aspects of T2DM.

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However, the effect on phasic dopamine responsiveness and insulin sensitivity in T2DM patients still needs to be studied. Gaining insight in these effects of bromocriptine could contribute to a novel treatment for T2DM. This could be efficient since current treatments show to have side-, no-, or a modest effect (18,19).

Recent studies have also found an effect of dopamine release on reward control. An increase in threshold of brain stimulation reward (BSR) procedure was found in obese rats as a result of striatal D2R expression knockdown in the dorsolateral striatum (20). This BSR procedure is executed by

actively obtaining rewarding electrical self-stimulation. Thus, more electrical self-stimulation has to be performed in order to reach the threshold for dopamine to acquire feelings of reward. In addition, brain imaging of obese humans showed that deficits in striatal D2R density contribute to food

reward hypofunction (21). Since feelings of reward in obesity are reached through food intake (22), these results indicate that striatal dopamine release is necessary for reward control and thus for controlling excessive feeding behavior.

In this study an attempt will be done to reinstate the circadian rhythm of dopamine in T2DM

patients by simulating the dopamine morning peak via a 12-week timed-bromocriptine therapy. The aim of this study is to assess the effect of the simulated dopamine morning peak on phasic

dopamine responsiveness, and whether improvement of responsiveness is associated with an increase in insulin sensitivity. In addition, the effect of simulated dopamine release on feeding behavior will be determined. It is hypothesized that in T2DM patients i. reinstatement of the dopamine morning peak by bromocriptine will restore phasic dopamine release to a certain extent, ii. the increase in phasic dopamine release will increase insulin sensitivity, and that iii. an increase in phasic dopamine release will improve excessive feeding behavior. Phasic dopamine release (measured as dexamphetamine-induced dopamine release) will be assessed via SPECT imaging, and insulin sensitivity (measured as exogenous glucose infusion rat) via a two-step

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hyperinsulinemic, euglycemic clamp. In addition, feeding behavior will be determined via neuropsychological questionnaires. These methods will be repeated after the intervention to

determine whether timed administration of bromocriptine had an effect. Based on previous studies, an increase in dexamphetamine-induced dopamine release and exogenous glucose infusion rate will be expected, as well as in controlling excessive feeding behavior.

2. Material and methods

2.1. Subject

One male Caucasian participant (69 years old) was recruited into the present study based on a screening in which the inclusion and exclusion criteria (see appendix 1) were checked for. This was verified by an interview to check for medical history, by a physical examination, by collection of a fasting blood sample to determine plasma liver enzyme, thyroid stimulating hormone and creatinine and by making an ECG at rest and during an exercise stress test to assess cardiac health.

Furthermore, the subject had to fill in neuropsychological questionnaires related to feeding behavior, as seen below. All procedures were approved by the Academic Medical Centre medical ethics committee and the subject provided written informed consent in accordance with the Declaration of Helsinki.

2.2. Two-step hyperinsulinemic, euglycemic clamp

After an overnight fast, the body composition was measured using BodPod (percentage fat mass and lean mass) whereafter the subject was admitted to the metabolic unit. Two catheters were inserted; one into an antecubital vein for infusion of the tracer, insulin and glucose, the other into a contralateral hand vein for sampling of arterialized venous blood. Primed continuous infusion of the [6,6-2_H₂_{]glucose tracer (prime equals 100 minutes of continuous infusion; continuous 0.11}

µmol/kg•min) was started at T=-2.00 hours and was continued until the end of the clamp. After a tracer-equilibrium period of 1 hour and 50 minutes, basal sampling was done at 5-minute intervals

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for 10 minutes. At T=0.00 hours the insulin infusion of 20 mU/m2_{•min (Actrapid [100 IU/ml],}

Novo Nordisk B.V., Alphen aan den Rijn, Netherlands) was started. To maintain a constant plasma glucose level of 5 mmol/L and to minimize changes in isotopic enrichment, glucose (enriched with [6,6-2_H₂_{]glucose) was infused. Insulin infusion was increased to 60 mU/m}2_{•min at T=2.10 hours.}

During one clamp session, blood samples were drawn at T=1.50 and T=4.00 at 5-minute intervals for 20 minutes. Part of these samples were drawn for immediate determination of plasma glucose concentration, enabling possible adjustments in exogenous glucose infusion to be made to maintain a glucose level of 5 mmol/L. The rest of the samples was used for determination of stable isotope enrichment in plasma, glucoregulatory hormones and FFA. Furthermore, the resting energy

expenditure (REE) (kcal•kg-1_•day-1_{) was measured twice during the clamp session. Until completion}

of the clamp session, the subject was not allowed to eat.

2.2.1 Clamp analysis

The exogenous glucose infusion rate (mg/kg•min) at T=2.10 and T=4.20 hours prior to the intervention was compared to the infusion rate after the intervention. A high infusion rate means that a high concentration of exogenous glucose has to be infused per minute to maintain the 5 mmol/L blood glucose level, and thus that the adipose tissue, liver and skeletal muscle are sensitive to insulin.

2.3. SPECT scan

Striatal D2/3Ravailability was measured using a high-resolution SPECT system at the AMC

(InSpira system) with intravenous dexamphetamine infusion (23–27). Two scans were made, each taking approximately 60 minutes. The subject took his last dose of oral glucose lowering drugs the day before the scan. Furthermore, potassium iodine drops or tablets were taken the day before and the morning of the scan to reduce iodide uptake of the radioligand and the subject consumed a standardized breakfast. At T=0.00 hours an intravenous cannula was placed for intravenous

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injection of the selective D2/3receptor antagonist radioligand [123I]iodobenzamide (IBZM) (64 MBq

bolus followed by constant infusion of 16 MBq/hour for 5 consecutive hours). This was continued till the end of the SPECT session. The dose per [123_{I]IBZM SPECT session was 4.9 mSv (144}

MBq), given a total dose of 9.8 mSv which is within the NCS-recommended maximum dose for male subjects >50 years (risk category llb). At T=2.00 hours a baseline SPECT scan was made to assess availability of the D2/3 receptor. Dexamphetamine (dexamphetamine sulphate 0.3 mg/kg ideal

bodyweight) was administered intravenously at T=3.15 hours. Meanwhile, physiological responses were monitored using an ECG. A second SPECT scan was made at T=4.00 hours to assess changes in availability of the D2/3 receptor. The intravenous cannula was removed an hour later.

2.3.1. SPECT analysis

Dexamphetamine-induced dopamine release (∆D2/3RBPND) was calculated as the change in striatal

D2/3Rbinding potential (D2/3RBPND) of [123I]IBZM before and after dexamphetamine infusion.

Volumes of interest were drawn over the left and right striatum and over the occipital lobe. D2/3R

BPND was calculated as the sum of both right and left striatum specific ratios. ∆D2/3RBPND was

calculated as follows: ((D2/3RBPND post-dexamphetamine - D2/3RBPND

pre-dexamphetamine)/D2/3RBPND pre-dexamphetamine)•100%.

2.4. Bromocriptine intervention

The intervention started with a bromocriptine administration of 1.25 mg every morning within two hours of awakening. The dose was increased with 1.25 mg every week for the next 4 weeks until a dose of 5 mg was reached (titration phase). For the following 8 weeks, the subject continued the administration of 5 mg bromocriptine every morning (stable phase). During the 12-week

intervention, the subject was contacted by telephone every week to assess potential side-effects and adherence. Furthermore, the subject had to visit the AMC three times for assessment of side-effects via an interview, a physical examination, bodyweight measurement and adherence through pill

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count. For additional information of bromocriptine, refer to the SmPC of the Medicines Evaluation Board (MEB) (28).

2.5. Questionnaires

To compare aspects of feeding behavior after the intervention with measures obtained at baseline, the subject had to repeat the following questionnaires:

2.5.1. General Food Cravings Questionnaire-Trait (GFCQ-T)

21 items that focus on a general trait of food craving that is scored on a 6-point Likert scale, ranging from never or not applicable to always. The questionnaire is divided into four subscales; 1)

preoccupation with food, 2) loss of control, 3) positive outcome expectancy, and 4) emotional craving (29).

2.5.2. Dutch Eating Disorder Examination Questionnaire (EDEQ)

30 items that focus on behavior correlated with Anorexia Nervosa or Bulimia Nervosa. The

questionnaire is divided into four subscales; 1) restraint, 2) eating concern, 3) shape concern, and 4) weight concern (30).

2.5.3. Dutch Eating Behavior Questionnaire (DEBQ)

33 items to assess three distinct eating behaviors in adults. The questionnaire divided into three subscales; 1) emotional eating, 2) external eating, and 3) restrained eating. Besides the items, the subject is asked to answer some general questions (i.e. history of weight) (31).

2.5.7. Yale Food Addiction Scale 2.0 (YFAS)

35 items that identify eating habits in the past year similar seen in classic areas of addiction. Every question falls under a DSM 5 Substance-Related and Addictive Disorders (SRAD) symptom criterion or clinical impairment. The subject chooses out of eight frequency response options

ranging from never to every day. The scale is divided into 4 subscales; 1) no food addiction, 2) mild food addiction, 3) moderate food addiction and 4) severe food addiction (32).

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2.6 Statistical analysis

As the data was obtained from 1 subject (N=1), a statistical analysis could not be carried out. Therefore, a qualitative analysis will be done instead of a quantitative analysis. However, when the coronavirus will allow us to, an interim analysis will be performed on 10 subjects that have

completed the study. For this purpose, a R-script has been written (see appendix 2).

3. Results

3.1. Bodyweight/composition, abdominal circumference and REE

Changes in bodyweight/composition, abdominal circumference and REE of the subject after the intervention in comparison to baseline are summarized in table 1. The subject gained weight slightly during the 12-week bromocriptine intervention. Furthermore, the fat mass reduced, and the lean mass, the abdominal circumference and REE increased.

Table 1 --- Descriptive characteristics of the subject at baseline and after a 12-week bromocriptine intervention.

3.2. Insulin sensitivity

Changes in exogenous glucose infusion rate between measurements at baseline and postintervention are summarized in table 2. Infusion rates at T=2.10 and T=4.20 hours were compared for

determination of differences. After the bromocriptine intervention, the exogenous glucose infusion rate (mg/kg•min) was decreased from 1.50 to 0.5 at T=2.10 hours and from 5.25 to 3 at T=4.20 hours. Since a low infusion rate means that a low concentration of exogenous glucose was infused per minute to maintain the 5 mmol/L blood glucose levels, a decrease in infusion rate suggests an impairment in insulin sensitivity.

Bodyweight (kg)

Fat mass (%) Lean mass (%) Abdominal

circumference (cm)

Resting energy expenditure (kcal•kg-1_•day-1₎

Pre-intervention 93.307 38.7 61.3 109 1478

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Table 2 --- Exogenous glucose infusion rate (mg/kg•min) at baseline and after a

12-week bromocriptine intervention.

Time Pre-intervention Post-intervention

T=1.50 1.25 0.5 T=1.55 1.25 0.5 T=2.00 1.25 0.5 T=2.05 1.50 0.5 T=2.10 1.50 0.5 T=4.00 4.75 3 T=4.05 4.75 3 T=4.10 5.25 3 T=4.15 5.25 3 T=4.20 5.25 3

3.3. Dexamphetamine-induced dopamine release

Dopamine D2/3R binding of the occipital lobe and left and right striatum was obtained before

(scan 1) and after (scan 2) dexamphetamine infusion (table 3). Additionally, the striatal dopamine binding potential (D2/3RBPND) and dexamphetamine-induced dopamine release ((∆D2/3RBPND)

were calculated (table 4). A 5.76% decrease in dexamphetamine-induced dopamine release was seen after the bromocriptine intervention.

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Table 3 --- Striatal and occipital dopamine D2/3Rbinding before (scan 1) and after (scan 2) dexamphetamine

infusion, measured at baseline and after a 12-week bromocriptine intervention assessed through [123_{I]IBZM SPECT}

imaging.

Pre-intervention

Region name Voxels Specific ratio

Scan 1 Occipital Ctx. 8292 0.00 (nan)

Right striatum 1688 0.68 (-3.79)

Left striatum 1688 0.77 (-4.04)

Left striatum 1688 0.71 (-4.13)

Postintervention

Region name Voxels Specific ratio

Left striatum 1688 0.60 (-4.33)

Left striatum 1688 0.59 (-4.33)

Table 4 --- Dopamine D2/3Rbinding potential (D2/3RBPND) before and after dexamphetamine

infusion and dexamphetamine-induced dopamine release (∆D2/3RBPND), measured at baseline

and after a 12-week bromocriptine intervention.

3.4. Questionnaires

Total scores and subscores of the questionnaires are summarized in table 5. The subscores as well as the total scores of the GFCQ-T, EDEQ and DEBQ show not to be different after the bromocriptine intervention in comparison to before. Since no extremes between the subscales of these questionnaires have been found, there seems to be no indication for any of these subscale behaviors. However, it is striking that the subject scored high on the ‘Loss of control’ subscale of the GFCQ-T, as well as on ‘Restraint eating’ of the DEBQ, since these subscales show to be contradictory. Furthermore, the total score on YFAS does show to have reduced after the bromocriptine intervention. Before the intervention, the subject had a total score of 3 on the

Measure Pre-intervention Post-intervention

D2/3RBPND pre-dexamphetamine infusion 1.45 1.31

D2/3RBPND post-dexamphetamine infusion 1.30 1.25

Dexamphetamine-induced dopamine release (∆D2/3RBPND) (%)

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YFAS in comparison to 0 after the intervention. Nevertheless, the scores indicate that no food addiction was or is present.

Table 5 --- Total scores and subscores of the General Food Cravings Questionnaire (GFCQ-T),

Dutch Eating Disorder Examination Questionnaire (EDEQ), Dutch Eating Behavior Questionnaire (DEBQ) and Yale Food Addiction Scale 2.0 (YFAS) at baseline and after a 12-week bromocriptine intervention. Questionnaires Subscales Pre-intervention Post-intervention

GFCQ-T Preoccupation with food 2.33 2.33

Loss of control 2.83 2.83

Positive outcome expectancy 2 2

Emotional craving 2.25 2.25 Total score 2.38 2.38 EDEQ Restraint 0.2 0.2 Eating concern 0 0 Shape concern 0 0 Weight concern 0 0 Total score 0.05 0.05

DEBQ Emotional eating 2.07 2.07

External eating 2.8 2.8

Restrained eating 3.1 3.1

Total score 2.51 2.51

YFAS Total score 3 0

4. Discussion

The findings of this study show that an attempt in reinstating the circadian rhythm of dopaminergic activity by a 12-week bromocriptine intervention was accompanied by a negative difference in dexamphetamine-induced dopamine release. A decrease in insulin sensitivity of the adipose tissue, liver and skeletal muscle was established as a possible result. These results indicate that phasic dopamine release was decreased and the subject became less sensitive to insulin after the intervention in comparison to baseline. This is contrary to what was expected to be found. Furthermore, no differences in feeding behavior were found associated to changes in phasic dopamine release using the GFCQ-T, DBEQ, EDEQ and YFAS.

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The obtained results of diminished phasic dopamine release are inconsistent with the results of previous studies that demonstrated that an increase in the dopamine morning peak, as a result of timed-bromocriptine administration, reinstated the circadian rhythm of dopaminergic activity (33). Following this circadian rhythm, the increase in morning dopamine release was expected to cause a decrease in tonic dopamine release and therefore should have enhanced phasic dopamine release. A possible explanation for these conflicting results could be the time needed for bromocriptine to reach its maximum concentration (Tmax) in the blood. Evidence shows that the timing of

bromocriptine administration is crucial for its effectiveness since the body has its natural circadian peak of dopaminergic activity in the CNS clock area. When bromocriptine is taken at a time of day that is out of phase from this natural rhythm, the intervention can be less effective or even

ineffective (10). Previous studies looking into the effect of bromocriptine on dopamine release primarily based their results on data obtained with Cycloset; a fast-absorbing formulation of the ergot derivative bromocriptine mesylate (34). This is formulated as bromocriptine-QR (quick-release) which means that oral administration in the morning produces a short increase in CNS dopaminergic activities. However, this present study used the oral Parlodel bromocriptine

formulation (28). Research shows that the Tmax under fasting conditions is different for Cycloset and Parlodel; 45-60 minutes versus 1 to 3 hours (17,28). Thus, there is a difference in duration to reach the maximum concentration of bromocriptine in the blood and therefor to simulate the dopamine morning peak. A study in nondiabetic humans shows that the dopamine morning peak is normally present within 2 hours of awakening (35). However, since Parlodel takes approximately 1 to 3 hours to reach the maximum concentration of bromocriptine in the blood, the dopamine

morning peak was presumably simulated later than 2 hours after awakening, causing an increase in tonic dopamine release and a decrease in phasic dopamine release. Therefore, it is likely that the dopaminergic activity was reinstated out of phase from its natural rhythm. Given the obtained decrease in phasic dopamine release, the difference in time needed to reach the maximum concentration of bromocriptine between Cycloset and Parlodel shows to be a reasonable

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explanation. For this reason, it would be interesting to look further into the effect of the simulated dopamine morning peak by bromocriptine administration on phasic dopamine release and insulin sensitivity using Cycloset instead of Parlodel to prevent the peak from potentially being simulated during the tonic phase of the circadian rhythm of dopamine.

In addition, former studies have shown that bromocriptine QR-therapy has a positive effect on insulin sensitivity in T2DM patients; causing a reduction in glycated hemoglobin (HbA1C) (16,17), improvement of glycemic control (10) and glucose tolerance (16). Therefore, the decrease in insulin sensitivity that was seen in the present study is in conflict with previous results. Assuming Parlodel indeed increased the tonic dopamine release instead of the dopamine morning peak, this could also explain the decrease in insulin sensitivity. An increase in the morning peak, and therefore in phasic dopamine release, is namely assumed to be needed for improvement of insulin sensitivity shortly after awakening (10). Thus, a decrease in phasic dopamine could presumably be responsible for the diminished insulin sensitivity.

Another possible explanation for the fact that phasic dopamine release was not increased by the timed-bromocriptine intervention, is the percentage of absorbed administered bromocriptine by the blood. Research shows that ~65-95% of Cycloset is absorbed in the blood when taken orally in contrast to 28% of Parlodel (17). Thus, a difference is seen in the amount of bromocriptine taken up by the blood when using either Cycloset or Parlodel. A study in insulin-sensitive, glucose tolerant hamsters showed a two-third reduction in the dopaminergic peak when the hamsters were brought into the insulin-resistant, glucose intolerant state (36). By this means, the dopamine morning peak has to be increased by two-third to be simulated as the normal peak. Since only 28% of Parlodel shows to be absorbed by the blood, this two-third increase is unlikely to be accomplished and thus an increase in phasic dopamine release could not be realized.

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Furthermore, it should be taken into account that stress and changes in daily sleep (<5 or >9 hours) have a great impact on the CNS dopamine function (35,37). These factors show to cause a

diminished circadian dopaminergic morning peak. Since participating in a research and taking a medicine that has not been fully proved to repair your disease could cause anxiety, it could be possible that the subject experienced more stress during the intervention. This could have resulted in a decreased dopaminergic morning peak and therefore have counteracted the effect of

bromocriptine. Therefore, it would be interesting to look further into the possibility if stress and/or sleep deprivation could counteract the effect of timed-bromocriptine administration.

In conclusion, this study shows that 12-weeks of timed-bromocriptine administration via Parlodel tablets decreases both phasic dopamine release and insulin sensitivity. Additionally, no differences in feeding behavior have been found associated to changes in phasic dopamine release. Limitations of this study include the small sample size and as a result that only the effect on the male gender has been researched. However, despite these limitations, the obtained results emphasize the importance of the research question. Since these results show to be reversed with what was expected, it is crucial to continue this study with a bigger sample size to verify the effect of timed-bromocriptine administration via Parlodel on phasic dopamine release and insulin sensitivity. Gaining insight in these effects of Parlodel administration could prevent this treatment from being recommended to T2DM patients and therefore prevent these patients from possibly getting worse. In addition, this insight narrows down the possible treatments for T2DM accompanied with side effects or no effect at all (18,19), and therefore brings us closer to finding a good and consistent treatment for T2DM, counteracting the suffering from this disease.

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5. Acknowledgements

I would like to thank Jamie van Son for her accompaniment during my internship at the endocrinology department of the Amsterdam UMC (location AMC). Furthermore, I would like to thank Mireille Serlie and Taco Werkman for examining my thesis.

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Appendix 1. Inclusion and exclusion criteria

1.1 Inclusion criteria

In order to be eligible to participate in this study, a subject must meet all of the following criteria: - Overweight/obese (BMI>25.0 kg/m2) T2DM patient, treated with oral glucose lowering

medication (except for DDP4-inhibitors).

- Insulin resistance (fasting insulin >70 pmol/L), except for patients on SU-derivatives (fasting insulin is not a reliable marker for insulin resistance when patients are treated with an insulin secretagogue).

- Age 50-70 years

- Males and postmenopausal females: (history, amenorrhoea, elevated FSH) - Stable weight (<10% change in bodyweight for 3 months prior to assessments) - Ability to provide informed consent.

1.2 Exclusion criteria

A potential subject who meets any of the following criteria will be excluded from participation in this study:

- Any current somatic (except for stable obesity- or T2DM-related comorbidities) or psychiatric disorder

- Shift work

- Uncontrolled hypertension

- The use of excessive alcohol or recreational drugs - Smoking

- Any use of medication (including NSAIDs) except for lipid lowering, blood pressure lowering drugs and occasional use of paracetamol (less frequent than 2 days a week) - History of psychiatric disorder or drug- or alcohol abuse

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- History of the use of dexamphetamine or dopamine agonists - Abnormal ECG at rest or during the exercise stress test - Positive family history of sudden death

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Appendix 2. R-script for N=10

Due to the corona virus, we were not able to obtain enough data for a statistical analysis. However, an interim analysis will be performed to analyse the data when 10 subjects (N=10) have completed the study. One of the aims of this study is to determine differences in multiple parameters

(descriptive characteristics, exogenous glucose infusion rate, dexamphetamine-induced dopamine release and scores on questionnaires) as a result of the 12-week bromocriptine intervention. These obtained values of the 10 subjects before the intervention will be compared with values after the intervention using a paired T-test (parametric). If the data shows not to be normally distributed, a Wilcoxon signed rank test will be performed (nonparametric).

The second aim of this study is to determine if changes in dexamphetamine-induced dopamine release caused an improvement in the exogenous glucose infusion rate and feeding behavior. Therefore, correlation coefficients between these variables will be calculated using Pearson

correlation. If the data shows not to be normally distributed, Spearman Rank Order correlation will be performed.

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2.1 Changes in descriptive characteristics

2.1.1. Import table ‘Descriptive’ into R

The table will consist of 4 columns (subject, descriptive characteristic, pre-intervention and post-intervention) and 50 rows (values for bodyweight [1:10], fat mass [11:20], lean mass [21:30], abdominal circumference [31:40] and REE [41:50]).

2.1.2. Calculate mean and sd, check paired T-test assumptions and compute either a paired T-test (parametric) or Wilcoxon signed rank test (nonparametric)

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2.2. Changes in exogenous glucose infusion rate

2.2.1. Import table ‘Glucose’ into R

The table will consist of 4 columns (subject, time, preintervention and postintervention) and 20 rows (values for T=2.10 [1:10] and T=4.20 [11:20]).

2.2.3. Repeat same script for values of T=4.20 hours

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2.3. Changes in dexamphetamine-induced dopamine release

2.3.1. Import table ‘Dopamine’ into R

The table will consist of 4 columns (subject, time, preintervention and postintervention) and 30 rows (values for D2/3RBPND pre-dexamphetamine infusion [1:10], D2/3RBPND

post-dexamphetamine infusion [11:20] and post-dexamphetamine-induced dopamine release [21:30]).

2.3.3. Repeat same script for values post-dexamphetamine infusion and dexamphetamine-induced dopamine release

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2.4. Changes in scores on questionnaires

2.4.1. Import tables ‘GFCQ-T’, ‘EDEQ’, ‘DEBQ’ and ‘YFAS’ into R 4 tables will be imported.

Table 1, named ‘GFCQ-T’, will consist of 4 columns (subject, subscales/total, preintervention and postintervention) and 50 rows (values for Preoccupation with food [1:10], Loss of control [11:20], Positive outcome expectancy [21:30], Emotional craving [31:40] and Total score [41:50]).

Table 2, named ‘EDEQ’, will consist of 4 columns (subject, subscales/total, preintervention and postintervention) and 50 rows (values for Restraint [1:10], Eating concern [11:20], Shape concern [21:30], Weight concern [31:40] and Total score [41:50]).

Table 3, named DEBQ, will consist of 4 columns (subject, subscales/total, preintervention and postintervention) and 40 rows (values for Emotional eating [1:10], External eating [11:20], Restrained eating [21:30] and Total score [31:40]).

Table 4, names YFAS, will consist of 3 columns (subject, preintervention and postintervention) and 10 rows (values for Total scores [1:10]).

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2.5. Correlations between changes in dexamphetamine-induced dopamine release and changes in insulin sensitivity

2.5.1. Import table ‘DopaGlu’ into R

Changes in dexamphetamine-induced dopamine release compared to baseline (delta BPND) can be

compared to changes in exogenous glucose infusion rate compared to baseline (delta glucose). Therefore, the table will consist of 3 columns (subject, deltaBP and deltaglucose) and 10 rows (values for each subject).

2.5.2. Check Pearson correlation test assumptions and compute either a Pearson correlation test (parametric) or Spearman’s rank order correlation (nonparametric)

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2.6. Correlations between changes in dexamphetamine-induced dopamine release and changes in feeding behaviour (GFCQ-T, EDEQ, DEBQ and YFAS).

2.6.1. Import table ‘DopaGFCQT’ into R

Changes in dexamphetamine-induced dopamine release compared to baseline (delta BPND) can be

compared to changes in total scores on GFCQ-T compared to baseline (delta score). Therefore, the table will consist of 3 columns (subject, deltaBP and deltascore) and 10 rows (values for each subject).

2.6.2. Check Pearson correlation test assumptions and compute either a Pearson correlation test (parametric) or Spearman’s rank order correlation (nonparametric)