DAMGO-induced acute activation of the µ-opioid receptor in the nucleus accumbens augments the counterregulatory response to insulin-induced hypoglycemia in Wistar rats

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Luna van der Gun

11704241

Bachelor thesis Psychobiology

Under the supervision of: Laura Koekkoek MSc

La Fleur Research Group, Academic Medical Centre (AMC) Amsterdam

19/06/2020

DAMGO-induced acute activation of the

µ-opioid receptor in the nucleus

accumbens augments the

counterregulatory response to

insulin-induced hypoglycemia in Wistar rats

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DAMGO-induced acute activation of the µ-opioid

receptor in the nucleus accumbens augments the

counterregulatory response to insulin-induced

hypoglycemia in Wistar rats

Luna van der Gun

Abstract

Maintaining adequate blood glucose levels is essential for the function and survival of the Central Nervous System (CNS). Therefore, the brain senses glucose availability and tightly regulates endogenous glucose production and glucose uptake. A newly discovered candidate in the CNS control of systemic glucose metabolism is the nucleus accumbens (NAc), whose glucoregulatory effect may be mediated by the opioid system. Male Wistar rats were bilaterally infused with the µ-opioid receptor (MOR) specific agonist DAMGO in the NAc and were thereafter tested for effects on insulin sensitivity with an intravenous insulin tolerance test. Results indicate that DAMGO-induced activation of the MORs in the NAc does not alter net peripheral insulin sensitivity but does augment the counterregulatory response to insulin-induced hypoglycemia. The mechanism by which the opioid system in the NAc mediates the counterregulatory response to acute hypoglycemia remains to be elucidated and may involve action of the dopamine system. Although further research is required to establish the exact function of the MORs in the glucoregulatory effect of the NAc, a previously undiscovered effect of the MOR system in the NAc on peripheral insulin tolerance is described. Thereby, the foundation for research into the opioid effects on glycemic control and possibly for an association of these effects with the pathogeneses of metabolic disease has been laid.

Keywords: DAMGO, µ-opioid receptor, nucleus accumbens, counterregulatory response to

hypoglycemia, insulin tolerance test

1. Introduction

Glucose is an essential source of energy, that is required for the function and survival of all mammals. Specifically the brain, that in basal state already utilizes 60% of total body glucose and is limited in its capacity to store glucose, heavily depends on circulating glucose for its energy need. For humans, blood glucose levels (glycemia) should therefore always be maintained between 3.0 and 5.6mM (Watts & Donovan, 2010). Glycemia is the result of a tightly regulated balance between endogenous glucose production (EGP) and glucose uptake of bodily tissues. In order to maintain sufficiently high glycemia, respondence to events that challenge this balance (e.g. food intake and exercise) is required. Key in maintaining this balance are the glucoregulatory hormones, of which insulin and glucagon are the most well-known. In case glycemia rises, for example after a meal, insulin is secreted from the pancreatic β-cells. Systemic insulin then lowers glycemia through the stimulation of glucose uptake by peripheral tissues, such as adipose, skeletal muscle and liver tissue ( Wolfe, 2007). Subsequently, peripheral tissues are provided with glucose supply for their energy need and excessive glucose is stored as glycogen in the liver. Additionally, hepatic glucose production (HGP) is inhibited by insulin (Sonksen & Sonksen, 2000). However, when glucose levels drop, the release of counterregulatory hormones such as glucagon, epinephrine, cortisol, and growth hormone is increased. These agents increase glycemia through the stimulation of HGP (Wolfe, 2007).

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Central nervous system (CNS) in control of systemic glucose homeostasis

Advances in the field of neuroscience from mid 19th-century onwards have shed light on the role of the brain in the regulation of whole body glucose metabolism (Bernard, 1855; Lam, Chari & Lam, 2009). The brain receives and integrates nutrient-related signals (such as glycemia) and adiposity-related signals (such as insulin, which circulates in proportion to body fat mass (Bagdade, Bierman & Porte, 1976)). This enables the brain to sense both current glucose availability and body energy stores. In response to these afferent inputs, adaptive responses such as adjusted feeding behavior and innervation of autonomic nerves reaching glucoregulatory organs are activated to promote balance of the homeostatic system (Schwartz & Porte, 2005). Via the sympathetic and parasympathetic nerves, the brain is able to innervate the liver, skeletal muscle and adipose tissue and effectively influence endogenous glucose production and uptake (Lam et al., 2009; Sandoval et

al., 2009; Watts, 2014; Lundqvist et al., 2019). Additionally, the brain can influence glucose

homeostasis by innervating the pancreas, thereby orchestrating the release of glucoregulatory hormones insulin and glucagon (Bertrand et al., 1996; Tsutsumi et al., 2002).

Classically, key players recognized in the CNS regulation of systemic glucose homeostasis are the hypothalamus and the brainstem. Their anatomical position nearby the third and fourth ventricle, that contain cerebrospinal fluid, allow these areas to access nutrients and hormones (Lundqvist et al., 2019; Mortazavi et al., 2014). Both brain areas contain glucose-sensing neurons (i.e. neurons that adjust their activity in response to changing extracellular glucose), enabling them to sense levels of circulating glucose and respond accordingly (Marty, Dallaporta & Thorens, 2007). These neurons are either glucose excited (GE) or glucose inhibited (GI) (for reviews on hypothalamic glucose sensing neurons see Burdakov & Gonzalez, 2009 and Levin et al., 2004). Apart from the ability to sense levels of circulating glucose, these areas serve as relay centers for humoral and neural feedback from the periphery (Obici, Zhang, Karkanias & Rossetti, 2002b; Campfield et al., 1995; Lundqvist et al., 2019). All together, these inputs determine the outflow of the hypothalamus and brain stem to glucoregulatory organs, by which modulation of EGP, pancreatic hormone secretion and glucose uptake of peripheral tissues in response to glycemia is achieved (Lam, Chari, Rutter & Lam, 2011; Buijs et al., 2001; Sudo et al., 1991). Herein, numerous studies have implicated roles for the arcuate nucleus (ARC), ventromedial hypothalamus (VMH) and the lateral hypothalamus (LH), that are part of an interconnected network of hypothalamic nuclei that has dense projections to the brainstem (Lundqvist et al., 2019; Ruud, Steculorum & Brüning, 2017; Lam et al., 2011; Roh, Song & Kim, 2016; Myers & Olson, 2012; Verberne, Sabetghadam & Korim, 2014). The brainstem in turn projects to the pancreas to regulate endocrine pancreatic functions and projects to the liver, potentially to affect HGP (Berthoud, 2004; Buijs et al., 2001; Wu et al., 2004; Yi et al., 2010).

However, novel studies indicate that other brain areas have additional effects on systemic glucose homeostasis. Many of the limbic structures, studied most in relation to reward and addiction, also contain glucose-sensing neurons (Papp et al., 2007; Dodd et al., 2010; Nakano et al., 1986; Izumi et

al., 1994). A body of evidence suggests that the nucleus accumbens (NAc), one of those limbic

structures, is involved in the CNS control of whole body glucose homeostasis. Firstly, besides containing glucose-sensing neurons (Papp et al., 2007), the NAc’s anatomical position close to the lateral ventricle (Neto, Oliveira, Correia & Ferreira, 2008) suggests that the NAc is able to sense and respond to glycemic events. Secondly, studies in rats have revealed neural connections between the NAc and the pancreas (Buijs et al., 2001) and between the NAc and the liver (Diepenbroek et al., unpublished), indicating the NAc may affect regulate endocrine pancreatic functions and HGP. Thirdly, electrical stimulation of the NAc using deep brain stimulation (DBS) has been found to influence glucose metabolism. A study of Diepenbroek et al. (2013b) revealed that DBS of the shell of the NAc (sNAc) upregulated systemic concentrations of glucose and glucagon in a region- and

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intensity-dependent manner in rats. In human studies as well, DBS in the area located at the border of the NAc core and ventral anterior limb of the internal capsule (vALIC) resulted in a change in glycemia (ter Horst et al., 2018). Electric stimulation along the vALIC was associated with increased insulin sensitivity of hepatic, adipose and muscle tissue and increased suppression of EGP and glucose disposal. Despite the ambiguity in the observed effects of NAc-DBS on glycemia, that are likely to be caused by the numerous factors affecting the cellular response to DBS (e.g. exact position electrodes, stimulation strength and duration etc.), it can be concluded from these studies that a role for the NAc in glucose metabolism through the control of pancreatic and/or hepatic output and peripheral insulin sensitivity is indicated. Thus, several studies implicate an important role for the NAc in the brain glucose-sensing circuitry that regulates peripheral glucose dynamics. However, it remains to be elucidated which neurotransmitter systems and cellular pathways underly these effects.

Potential roles for dopamine and opioids in the effects of the Nucleus Accumbens on whole body glucose homeostasis

As it is known that, both in rats (Sesia et al., 2010) and humans (Figee et al., 2013), electrical stimulation of the NAc results in dopamine release, the scope shifted towards dopamine (DA) as the potential pivotal neurotransmitter in the glucoregulatory effects of NAc activation. Striatal dopamine signaling has been associated with glucose metabolism both in human (ter Horst et al., 2018) and in animal studies. An elegant, yet unpublished study of Diepenbroek et al. has found that in rats, altering dopamine transmission in the sNAc directly influences HGP via activation of the vagus nerve. A pharmacological increase of DA in the NAc, induced by the intra-NAc infusion of the dopamine reuptake inhibitor vanoxerine, resulted in decreased EGP and a subsequent decrease in glycemia. As these observations were accompanied by increased vagal activity and hepatic vagotomy prevented the vanoxerina-sNAc-induced decrease in EGP, a neural route of the NAc to the liver via the nerves vagus was proposed.

Besides DA, opioids – inhibitory neurotransmitters most researched in relation to pain signaling (Zubieta et al., 2001) – are abundantly released within the NAc (Mansour et al., 1988) and may also mediate the glucoregulatory effects of the NAc. In mice, acute intracerebroventricular (i.c.v.) infusion of the µ-opioid receptor (MOR) with the selective MOR-agonist (D-Ala2, NMe-Phe4, Gly-ol5)-enkephalin (DAMGO) impaired glucose tolerance in a dose-dependent manner (Tuduri et al., 2016). This effect was attributed to a DAMGO-induced hampered insulin secretion from the pancreas in response to an increase in plasma glucose. Additionally, i.c.v. DAMGO administration was found to impair insulin tolerance and increase HGP. Thus, as pharmacological stimulation of MOR has been shown to affect whole body glucose homeostasis and MORs are widely distributed throughout the NAc (Mansour et al., 1988), the MOR system was hypothesized to be involved in the glucoregulatory function of the NAc. In order to research the potential involvement of the MORs in the NAc control of glycemia, the current work tested the effect of intra-NAc DAMGO-infusion on insulin sensitivity in male Wistar rats using a classic intravenous insulin tolerance test.

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2. Methodology

Animals

Male Wistar rats (240-280g) (Charles River, Sulzfeld, Germany) were housed 7 days before surgery in groups of four per cage to acclimatize to their new environment (temperature = 21,5±0,8°C, humidity = 60±2%, light-controlled room with a 12/12 h light-dark schedule (lights on at 7:00am hours), cage enrichment consisted of gnawing stick and PVC shelter). Rats were introduced to human handling in the period prior to surgery. All animals had ad libitum access to standard laboratory chow (Teklad global diet 2918, 18,6% protein, 44,2% carbohydrate, and 6,2% fat, 3,1 kcal/g, Envigo) and tap water prior to surgery and testing. After surgery, rats were individually housed in Plexiglas cages (25 x 25 x 35 cm). Herein, cage enrichment consisted of a gnawing stick solely. The experiment was consecutively performed on two groups of animals of which eight animals were excluded from the study due to untimely death or post-surgical complications that hindered further experimenting, resulting in a combined total of 24 animals. Additionally, animals whose cannulas in retrospect turned out to be misplaced outside of the NAc were excluded from the analysis (n=6). The experiment was approved by the Committee for Animal Experimentation of the Netherlands Institute for Neuroscience, Netherlands.

Procedure

Surgery

Wistar rats were initially anesthetized by the intraperitoneal injection of 80 mg/kg ketamine, 8.0 mg/kg xylazine and 0.1 mg/kg atropine. Animals were continuously assessed for anesthetic depth by firm toe pinches throughout the surgery. Marked increase in respirations or reflex were considered indicators of lack of anesthesia (JoVE Science Education Database, 2020), after which ketamine was injected intraperitoneally. To enable insulin infusion and extraction of blood samples for testing, all animals were implanted with an intra-arterial silicone catheter in the jugular vein, according to the method of (Steffens, 1969). To prevent diffusion of blood into the catheter, causing blockage by coagulation, a solution of polyvinylpyrrolidone (PVP) in heparin was injected into the catheter after surgery and thereafter renewed twice a week. Additionally, the animals were bilaterally implanted with 8 mm cannulas aimed at the sNAc (co-ordinates relative to bregma, lateral: +2.8 mm, A/P: +1.4 mm, ventral: -7.1 mm from the skull, cannulas are placed in an angle of 10° in the frontal plate), using a stereotaxic apparatus (Kopf, models 902 and 942). During brain surgery, lidocaine was directly applied on the skull for local anesthesia. Cannulas were occluded by stainless steel dummy cannulas. Catheters and cannulas were fixed on the skull with four anchor screws and dental cement. All animals received 0.3 mL of 2.5 mg/mL carprofen as pain medication and approximately 4 mL NaCl by subcutaneous injection during surgery and another dose of carprofen the day after. Directly after surgery, rats were placed in an incubator until consciousness was regained to prevent hypothermia. Thereafter, they were moved into their individual housing. The rats received a recovery period of 7 days, during which they were handled daily to minimize stress. Each day, body weight of the animals was measured to assess recovery of the surgery and post-operational blood clots were removed when estimated to be painful for the animal or obstructing for further experimenting. All animals were moved into clean cages once a week.

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Figure 1. Schematic representations of aims of surgical intervention. (A) Schematic representation of cannula

placement. All animals were bilaterally implanted with cannulas aimed at the sNAc, using a Kopf stereotaxic apparatus. (B) Schematic representation of intra-arterial silicone catheter in the jugular vein according to the method of (Steffens, 1969).

Drug delivery by intra-cerebral cannula

Infusion procedure effects were tested prior to experimental DAMGO/vehicle infusion by infusing phosphate-buffered saline (PBS) and thereafter checking for weight loss and remarkable feeding behavior. None of the rats responded peculiar to PBS infusion. Each animal was assigned to a treatment condition (DAMGO or vehicle) based on body weight. DAMGO-animals received 0.25 µg DAMGO resolved in 0.3µL sterile 0.9% PBS, control-animals received vehicle-infusion with 0.3 µL sterile 0.9% PBS. Bilaterally, the 300 nL infusate was administered to the brain with 0.3 µL/min via an internal injector that surpassed the cannula by 0.2mm and was attached to an infusion pump.

Intravenous Insulin Tolerance Test (ivITT)

All animals were back at or over preoperational body weight at the time of the ivITT. Animals were connected to a chain in order to release potential force on the catheter during head movements. Chains were introduced to the animals 24h before testing to minimize stress effects on glycemia. Animals had limited availability of chow (20,0 g) in the night before the ivITT to achieve fasting state. Glycemia was measured once approximately five minutes before (t=-5) and at six time points after drug delivery (t=0, t=5, t=10, t=20, t=30, t=60), using a custom glucose meter (Freestyle Freedom Lite, Abbot, Hoofddorp, the Netherlands). Directly after the t = 0 measurement, a shot of 0,1 IU/kg insulin was injected into the jugular vein.

Histology

At the end of the experiment animals were anaesthetized with CO2/O2 mixture (6:4) followed by 70%

CO2 and sacrificed by decapitation. Brains were then rapidly removed, frozen on dry ice and stored at

-80°C. Brain tissue was cut on a Leica CMI950 cryostat in 35 µm sections at -17°C. For verification of cannula placement, slides were Nissl-stained and examined with a microscope to determine the location of the cannulas. Herein, tissue damage that was attributed to the cannula or injector and the expected diffusion distance of the infusate were taken into consideration. Using the rat brain atlas,

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location of the infusate was estimated for each cannula. For each cannula within the NAc, the subregion (shell or core) of infusion and location (rostral or caudal) relative to bregma+1.28 (median of coordinates of cannulas of intra-NAc infused animals) was determined, providing categories for later additional analyses of within-NAc effects of DAMGO-treatment.

Statistical analysis

Data analysis was performed using Excel, Rstudio version 3.4.1. and GraphPad Prism version 8. To test if groups differed prior to the experiment (measured at t = -5) a two-tailed Mann-Whitney U test was performed. All experimental measures of glycemia were compared to baseline (t = 0). Comparisons of Δglycemia over time between DAMGO- and vehicle-treated animals were tested for significance using a two-way RM ANOVA with Geisser-Greenhouse correction. As the RM ANOVA is robust to violations of normality, normality of the Δglycemia-data of each of the treatment groups was assumed at all time points. By the use of the Geisser-Greenhouse correction, sphericity was not assumed. Sidak’s multiple comparisons test was used to compare differences between the two treatment groups in Δglycemia at each time point. For all animals, areas under the ITT-curve were calculated for negative peaks (indicative of insulin sensitivity) and for positive peaks (indicative of the counterregulatory response to acute hypoglycemia) separately. Assumptions of an unpaired t-test were examined using Shapiro-Wilk tests of normality and the Levene’s test for homogeneity of variance. Thereafter, negative peak areas and positive peak areas were tested for significant difference between treatment groups using a two-tailed unpaired t-test and an one-tailed1_{Mann-Whitney U test respectively.}

Data of DAMGO-treated animals whose cannulas were both misplaced in the ventral pallidum (VP) was compared to that of the DAMGO- and vehicle-treated animals that were included in the study (and whose cannulas were considered successfully aimed at the NAc) using two-way RM ANOVA (with Geisser-Greenhouse correction) and post-hoc Tukey’s multiple comparisons test. One-way ANOVA and Kruskal-Wallis test were used for negative and positive peak analyses respectively, based on the results of the using Shapiro-Wilk tests of normality and the Bartlett test of homogeneity of variance. Tukey’s and Dunn’s multiple comparisons test were employed for post-hoc analyses.

Additional analyses were performed on the data to test for effects of cannula placement within the NAc, either between shell and core or over a rostral-caudal gradient, on the treatment effect of DAMGO. In order to do investigate if intra-NAc DAMGO effects originate from the shell(sNAc), DAMGO-treated animals were subdivided into two groups based on the estimated amount of infusate in the sNAc (one or two cannulas aimed at the sNAc). To study rostral-caudal effects of intra-NAc DAMGO-infusion, DAMGO-treated animals with both cannulas located anterior to bregma+1.28 were assigned to the subgroup with more rostral DAMGO-effects, whereas animals with both cannulas located posterior to bregma+1.28 were assigned to the caudal-NAc subgroup. For both analyses, the effect of vehicle-treatment was assumed not to depend on cannula placement and DAMGO-treated animals that could not be assigned to a subgroup due to the inability to accurately determine precise cannula placement or deviations from the inclusion criteria of the groups were excluded. For both analyses, two-way RM ANOVA (with Geisser-Greenhouse correction) and Tukey’s post-hoc comparisons test were performed. Based on the results of the using Shapiro-Wilk tests of normality and the Bartlett test of homogeneity of variance, positive and negative peak areas were tested parametrically using one-way ANOVAs, except for the positive peak areas of the rostral-caudal NAc-analysis that required a Kruskal-Wallis test. Tukey’s multiple comparisons tests were employed for post-hoc analysis of significant ANOVAs.

1 Based on the ITT-graph, an accurate expectation of the difference in positive peak area could be formed, that allowed for one-tailed statistical testing.

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3. Results

In advance, the treatment groups did not differ in basal glycemia (p-value>0.05)(Fig. 2b). However after drug delivery, the glycemic response to insulin significantly differed between DAMGO-treated and vehicle-treated animals (F(5,80)=4.385, p-value<0.01). This significant difference could not be attributed to a certain time point, as was revealed with Sidak’s multiple comparisons test (all p-values>0.05)(Fig. 2c). Based on Fig. 2c the counterregulatory response to insulin-induced hypoglycemia, represented by the positive peak area, was presumed to be significantly greater in the DAMGO-treated animals than in the vehicle-treated animals. Indeed, the two treatment groups did not differ significantly in mean negative peak area (t(16)=0.580, p-value>0.05) (Fig. 2d), but did differ in positive peak area (U=19, p-value<0.05)(Fig. 2e).

Four animals were found to have both cannulas misplaced in the VP, rather than in the NAc. An examination of their ivITT-results lead to the speculation that DAMGO-infusion in this area also affects the counterregulatory response to insulin-induced hypoglycemia, possibly even more than in the NAc (Fig. 3c). Indeed, the glycemic response to insulin significantly differed between vehicle-treated animals, NAc-infused and VP-infused DAMGO-vehicle-treated (F(10,95)= 4.237, p<0.0001). Although Tukey’s multiple comparisons test revealed that the treatment groups did not differ significantly at any specific time point (all p-values>0.05), analyses of the negative peak area (F(2,19)=0.161, p>0.05) and positive peak area (Kruskal-Wallis statistic=7.505, p-value<0.05) show that the main difference between the groups is manifested in the counterregulatory response to insulin-induced hypoglycemia. Post-hoc Dunn’s multiple comparisons test revealed that only the positive peak area of the vehicle-treated group and the DAMGO in VP-group differ significantly (value<0.05; all other p-values>0.05)(Fig. 3e).

Based on earlier research, the glucoregulatory effect of the NAc was expected to manifest in the sNAc, rather than in the core of the NAc (cNAc) (Heimer et al., 1991). Additionally, the effect of DAMGO may vary across the rostral-caudal axis of the NAc (Salgado & Kaplitt, 2015). Therefore, additional analyses were performed on the DAMGO-treated animals. In both cases, the groups did not differ in basal glycemia before treatment (p-values>0.05)(Supplemental Fig. S1ae). Animals treated with vehicle-, unilateral or bilateral intra-sNAc DAMGO-infusion, significantly differed in their glycemic response to insulin (F(10,60)=2.481, p-value<0.05). However, animals with DAMGO unilateral and bilateral sNAc-infusion only significantly differed at t = 20 (value<0.05; all other p-values>0.05)(Supplemental Fig. S1b). Mean negative peak areas did not significantly differ (F(2,12)=1.488, p-value>0.05)(Supplemental Fig. S1c). Positive peak areas only differed significantly between the vehicle-treated group and the unilateral DAMGO sNAc-infusion group (p-value<0.01; all other p-values>0.05)(Supplemental Fig. S1d). For the analysis of DAMGO-effects over the rostral-caudal axis of the NAc, two-way RM ANOVA revealed significant differences in glycemic response to insulin between the groups (F(10,65)=2.867, p-value<0.01)(Supplemental Fig. S1f). However, no differences in glycemic response to insulin were found between DAMGO-treated animals with a more rostral NAc-infusion and DAMGO-treated animals with a more caudal NAc-infusion (all p-values>0.05) (Supplemental Fig. S1gh).

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Figure 2. Bilateral DAMGO-infusion in the nucleus accumbens does not alter peripheral insulin sensitivity but does augment the counterregulatory response to insulin-induced hypoglycemia in Wistar rats. (A) Schematic

representations of cerebral cannula for drug delivery aimed at the NAc. (B) Box plots of basal glycemia of the groups, measured prior to the ivITT (t=-5). (C) Treatment effect of bilateral intra-NAc DAMGO-infusion on glycemia following an intra-arterial injection of insulin (at t=0) over time. Values are represented as mean ± SEM. (D) Bar graph of the negative peak areas of the ivITT-graphs in figure 2c. Values are represented as mean ± SEM. (E) Box plots of the positive peak areas of the ivITT-graphs in figure 2c. Box displays the median and interquartile range, whiskers show minimum and maximum. *p< 0.05, ns = non-significant.

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Figure 3. Effects of bilateral DAMGO-infusion in the ventral pallidum with respect to the counterregulatory response to insulin-induced hypoglycemia may concede those of DAMGO-infusion in the nucleus accumbens.

(A) Schematic representation of misplaced cannulas aiming at the VP rather than at the NAc. (B) Bar plot of basal glycemia of the groups, measured prior to the ivITT (t=-5). (C) Treatment effect of bilateral intra-NAc and intra-VP DAMGO-infusion on glycemia following an intra-arterial injection of insulin (at t=0) over time. Values are represented as mean ± SEM. (D) Bar graph of the negative peak areas of the ivITT-graphs in figure 3c. Values are represented as mean ± SEM. (E) Box plots of the positive peak areas of the ivITT-graphs in figure 3c. Box displays the median and interquartile range, whiskers show minimum and maximum. *p< 0.05, ns = non-significant.

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4. Discussion

The effect of DAMGO-infusion in the NAc on glycemia over time after an insulin bolus was found to be significantly different from that of vehicle-infusion. However, no significant difference between DAMGO-treated animals and vehicle-treated animals was found in the initial drop in glycemia directly after the insulin bolus, as illustrated by the negative peak area (Fig. 2d). The rise in glycemia after this insulin-induced hypoglycemia however was significantly different be DAMGO-treated animals and vehicle-treated animals, as shown by the positive peak area (Fig. 2e). Thus, based on these results it can be concluded that DAMGO-induced activation of the MOR in the NAc does not alter net peripheral insulin sensitivity but does augment the counterregulatory response to insulin-induced hypoglycemia in Wistar rats. The augmented counterregulatory response suggests DAMGO in the NAc affects the secretion or sensitivity to one or more glucoregulatory hormones (glucagon, epinephrine, glucocorticoid and growth hormone). Which one(s) remains to be elucidated and is already under investigation of the la Fleur research group.

The observed effect of DAMGO-treatment on glycemia following insulin injection is in contrast with the results of Tuduri et al. (2016), who concluded that central DAMGO-administration acutely impaired insulin sensitivity. Remarkably, they found glycemia in DAMGO-treated mice to continue to rise up until approximately 20 minutes after insulin was injected. However, both the glycemia of the DAMGO- and vehicle-treated mice did not return to baseline within 120 minutes after insulin injection. This indicates that the animals had abnormally high glycemia prior to insulin injection, likely to be caused by stress effects, that are generally higher in mice than in rats (Kent Scientific Corperation, 2019). Thus, discrepancies in the ITT results may be explained by methodological differences. Additionally, as the DAMGO was administered i.c.v. in the study of Tuduri et al., it is likely that it affected multiple areas anatomically located close to ventricular sites, whereas the current study specifically targets the NAc.

Based on the results of the current ITT, it may be speculated that DAMGO could have induced an increase in basal glycemia (independent of the insulin-induced hypoglycemia) (Van Loon & Appel, 1981) that only started to occur at t = 20, when the data of the DAMGO-treated animals started to differ from that of vehicle-treated animals. However, unpublished data of the research group of La Fleur, acquired in the period prior to my internship, shows that intra-NAc DAMGO-infusion does not affect basal glycemia (Supplemental fig. S2a). In another cohort of Wistar rats that were infused 20 minutes prior to insulin injection, no effects of DAMGO-treatment were found on the glycemic response (Supplemental fig. S2b). Thus, the effects of DAMGO on the glycemic response to insulin-induced hypoglycemia observed in the current study are rightfully attributed to influences of DAMGO on the counterregulatory response to insulin-induced hypoglycemia.

Although DAMGO-effects were specific to the NAc, the precise mechanism of action that eventually leads to an altered glycemic response to insulin remains unknown. MORs are distributed throughout the NAc (Mansour et al., 1988) and as the NAc is targeted as a whole, it is unclear which type(s) of intra-NAc cell populations are involved in the observed effects on glycemic control. The NAc contains D1- and D2-expressing medium spiny neurons, GABAergic interneurons and cholinergic interneurons, that all respond differently to MOR activation and result in different effects on the net outputs of the NAc. Thus, the amount of possible circuitries by which DAMGO-infusion in the NAc eventually leads to alterations in the response to insulin-induced hypoglycemia is countless and further research is required to reveal the network involved in the DAMGO-induced NAc effects on glycemia.

The circuitry may or may not involve the action of additional neurotransmitters within the NAc. The glucoregulatory effect of intra-NAc DAMGO-infusion may be mediated by DA, whose action in the

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NAc has already been associated with glucoregulation (Diepenbroek et al., unpublished). Several animal studies show that pharmacological stimulation of MOR with DAMGO in the NAc leads to significantly elevated levels of extracellular concentrations of DA and its metabolites (Spanagel et al., 1990; Kalivas & Duffy, 1990). In addition to a DAMGO-mediated increase in extracellular DA, DAMGO might also affect the efficiency of DA-molecules in the excitation of medium spiny neurons. DAMGO has been shown to increase the intrinsic excitability of striatal D1- and D2-expressing medium spiny neurons, especially in the ventromedial striatum that the NAc is part of (Ma et al., 2012). The increase in intrinsic excitability of the medium spiny neurons is, by definition, caused by an alteration in the number and distribution of ion channels and receptors that contribute to the depolarization potential. This suggests DAMGO initiates a cascade of cellular processes leading to increased efficiency of excitatory neurotransmitters, such as DA. Although these studies implicate that MOR stimulation affects DA neurotransmission in the NAc, it remains to be elucidated if the effects of MOR stimulation on NAc glucoregulation are DA-mediated. A synergy of opioids and DA in glucoregulatory function of the NAc is not unlikely, but independent mechanisms or a combination of DA-dependent and -inDA-dependent mechanisms cannot be ruled out. To study the potential synergy between DA and opioids in the NAc control of glycemia, DA action could be blocked by infusing D1- & D2-antagonists in the NAc prior to intra-NAc DAMGO- or vehicle-infusion in a follow-up ivITT-study. Herein, the addition of conditions in which the MOR system is pretreated with the infusion of the selective and irreversible MOR antagonist β-FNA and subsequently the DA system is upregulated in the NAc (e.g. with vanoxerine-infusion) may yield interesting results that can add to a more complete picture of the contributions and dependencies of DA and opioids in the glucoregulatory function of the NAc .

DAMGO-infusion in the NAc eventually results in an augmentation of the counterregulatory response, but what brain areas and descending nerves are part of the trajectory that is responsible for this effect remains to be elucidated. The ventromedial hypothalamus is well known to be involved in the counterregulatory response to hypoglycemia (Borg et al., 1994). However, evidence of a functional connection from the NAc to the ventromedial hypothalamus is sparse (Canteras, Simerly & Swanson, 1994). Regardless, as hypothalamic nuclei are strongly interconnected, NAc outputs may eventually reach the ventromedial hypothalamus via other hypothalamic nuclei. The NAc is predominately connected to the lateral hypothalamus (LH) (Powell & Leman, 1976; Sano & Yokoi, 2007), that is known to be involved in the control of systemic blood glucose concentrations and EGP (Yi et al., 2009; Yi et al., 2010). The connection between the NAc and the LH has extensively been researched in relation to reward-related feeding, but may additionally be involved in glucose homeostasis independent of food intake. The potential involvement of the LH in the glucoregulatory effect of the NAc was examined in two of the previously described studies of Diepenbroek et al. The previously described upregulations of plasma glucose and glucagon induced by DBS in the NAc were associated with increased activation specifically of the LH, as shown with c-Fos expression (Diepenbroek et al., 2013b). Furthermore, Diepenbroek et al. found that alterations in glycemia induced by vanoxerine-infusion in the sNAc were mediated by an increased GABAergic transmission from the sNAc to the hypothalamus. Blockage of the GABAergic neurotransmission from the sNAC to the LH by the infusion of the GABA-antagonist bicuculline into the LH prior to the vanoxerine-NAc-infusion prevented the vanoxerine-induced reduction of glycemia. Diepenbroek et al. concluded that the projections from the sNAc to the LH are likely part of a NAc-LH-vagus-liver axis that is involved in the DA-mediated glucoregulatory effects of the sNAc (Diepenbroek et al., unpublished).

Direct GABAergic projections from the NAc to the LH originate from the medium spiny neurons (Stratford & Kelley, 1999). However, the GABAergic output of the NAc additionally is able to affect the LH via a projection to the VP. Medium spiny neurons of the sNAc tonically suppress the VP (Zahm &

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Heimer, 1990), that in turn inhibits the LH (Groenewegen, Berendse & Haber, 1993). Thus, DAMGO-induced inhibition of the neurons of the NAc may either result in net inhibition or in disinhibition of the LH. Interestingly, animals whose cannulas were misplaced in the VP exerted DAMGO-induced effects on the glycemic response to insulin similar to those observed in animals infused with DAMGO in the NAc. Although no significant difference was found between NAc-infused and VP-infused DAMGO-treated animals, the trend in the results allow for the speculation that DAMGO-infusion in the VP results in an even higher rise in glycemia after insulin-induced hypoglycemia than when infused in the NAc (Fig. 3ce). This suggests that the DAMGO-induced effects on counterregulatory response result from a disinhibition of the LH, either by DAMGO-induced inhibition of the NAc or by DAMGO-induced inhibition of the VP. However, as this speculation is based on the results of a small cohort of animals with misplaced cannulas, further testing is needed to validate the current observations regarding the role of the VP in glycemic control. The NAc may modulate (either via the VP or directly) hypothalamic nuclei that affect systemic glucoregulation. However, via direct projections to the periaqueductal gray (Salgado & Kaplitt, 2015), a major player in autonomic function that has been hypothesized to be vital to the counterregulatory response (Fioramonti, Song, Vazirani, Beuve & Routh, 2011), the NAc may also be able to influence glycemia independent of hypothalamic influences.

The efferent projections of the NAc affected by DAMGO-infusion may depend on the precise site of infusion within the NAc. Efferent connections and the distribution and density of neurotransmitter receptors differ between the sNAc and the cNAc (Heimer et al., 1991, Salgado & Kaplitt, 2015). Additionally, both in terms of structure and function, differentiation within the NAc was identified over a rostral-caudal axis (Salgado & Kaplitt, 2015). Therefore, it was speculated that intra-NAc differences in cannula placement could explain inter-subject variation of DAMGO-infused animals. However, additional analyses regarding the area of DAMGO-infusion within the NAc did not yield results that confirm the hypothesis that the amount of DAMGO-infusate in the shell of the NAc determines its effect size, nor that it matters whether DAMGO is infused more rostrally or caudally within the NAc. Notably, although positive and negative peak areas did not depend on the amount of DAMGO-infusate in the sNAc, the initial drop in glycemia induced by the injection of insulin was significantly greater in animals with bilateral DAMGO-infusion in the sNAc than in vehicle- and unilateral sNAc-DAMGO-treated animals (at t = 10). Based on these results, it is speculated that DAMGO may affect insulin sensitivity when enough of the infusate reaches the sNAc.

However, limitations of the present work should be taken into account when interpreting the results. Firstly, as the assessment of cannula placement is a reasonably gross manner of estimating the area of infusion, the selection of animals for each treatment group may be somewhat inaccurate. Especially for the additional analyses performed on intra-NAc differences, the low spatial resolution of cannula placement assessment may have affected the outcome of the analyses. Secondly, the small sample sizes increase the probability of type II errors. Thirdly, the statistical analyses performed in this study assume that subjects have no influence on each other. However, as all rats were in the same room during experimenting and rats produce species-specific vocalizations that have the capability of changing the emotional state and subsequently the glycemia of the receivers (Brudzynski, 2013; Chang et al., 2013), complete independency of the animals may be questioned. Lastly, although treatment groups were handled similarly and thus should have experienced equal amounts of stress, there is some evidence that suggests stress effects on glycemia may have differed between the treatment groups. In mice, endogenous opioids were found to mediate stress-induced hyperglycemia (Amir & Bernstein, 1982). Whether DAMGO could have augmented the hyperglycemic effects of stress in the present work is however unclear.

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Although the current study does not specifically address the physiological states in which the MORs of the NAc are activated, the present data strengthen the evidence that the NAc is involved in systemic glucoregulation. Moreover, a previously undiscovered role for the MOR system of the NAc in the counterregulatory response to acute hypoglycemia was described. These results highlight the importance of further research into the role of brain areas other than the classically recognized hypothalamic nuclei and brain stem in systemic glucose control. A more thorough understanding of the role of the brain in glycemic control, specifically in insulin sensitivity, might contribute to the battle against the ongoing obesity epidemic and closely related upsurge in type II diabetes (Blüher, 2019; Parvez Hossain et al., 2007). Especially since the opioid system in the NAc is strongly associated with the non-homeostatic feeding that causes obesity-related type II diabetes (for an excellent review on the role of the opioid system in the NAc in reward-related aspects of feeding see Kelly et al., 2005), the insights derived from the current study may eventually add to a deeper understanding of the pathogenesis of obesity-related type II diabetes. Finally, the insight that the opioid system affects neuroendocrine control of glucose homeostasis could provide a novel potential target for pharmacotherapies for metabolic diseases such as diabetes mellitus type II.

However, application of the newly acquired knowledge regarding the opioid system of the NAc in systemic glucoregulation requires more insight into the neural mechanism underlying the observed DAMGO effects. Further research should investigate the potential involvement of other neurotransmitter systems, such as the DA system. Additionally, although small sample sizes and low spatial resolution of cannula placement examination limited the interpretation of the present results regarding intra-NAc differences in the glucoregulatory effect of DAMGO, these results illustrate the need for further research into the specific involvement of intra-NAc subregions in the glycemic response to insulin.

Thus, although further research is required to examine the precise role of the MOR in the glucoregulatory function of the NAc, the present work describes a previously undiscovered effect of the opioid system in the NAc on peripheral insulin tolerance, specifically of the counterregulatory response to insulin-induced hypoglycemia. By doing so, the foundation for research into the opioid effects on glycemia and possibly for an association of these effects with the pathogeneses of metabolic disease has been laid.

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Supplement

S1. Details of statistical analyses

Test statistic p-value

DAMGO- & vehicle-infusion in the NAc

Sample size vehicle-treated animals n=10

Sample size DAMGO-treated animals n=8

Basal glycemia (t = -5)

Median ± IQR vehicle-treated animals 4.5±0.2 Median ± IQR DAMGO-treated animals 4.4±0.25

Shapiro-Wilk Test of Normality vehicle-treated animals W=0.953 p>0.05 Shapiro-Wilk Test of Normality DAMGO-treated animals W=0.821 p=0.048 Levene’s Test for Homogeneity of Variance L(1,15)=0.197 p>0.05 Two-tailed Mann-Whitney U Test U=29.50 p>0.05

Two-way RM ANOVA F(5,80)=4.385 p=0.001

Geisser-Greenhouse’s epsilon epsilon = 0.609 Sidak’s multiple comparisons test

t = 0 p>0.05 t = 5 p>0.05 t = 10 p>0.05 t = 20 p>0.05 t = 30 p>0.05 t = 60 p>0.05

Negative peak area

Mean ± SEM vehicle-treated animals 13.962±2.429 Mean ± SEM DAMGO-treated animals 12.039±2.114

Shapiro-Wilk Test of Normality vehicle-treated animals W=0.960 p>0.05 Shapiro-Wilk Test of Normality DAMGO-treated animals W=0.878 p>0.05 Levene’s Test for Homogeneity of Variance L(1,16)=0.066 p>0.05 Two-tailed unpaired t-test t(16)=0.580 p>0.05

Positive peak area

Median ± IQR vehicle-treated animals 3.234±6.025 Median ± IQR DAMGO-treated animals 17.25±21.923

Shapiro-Wilk Test of Normality vehicle-treated animals W=0.813 p=0.021 Shapiro-Wilk Test of Normality DAMGO-treated animals W=0.921 p>0.05 Levene’s Test for Homogeneity of Variance L(1,16)=12.181 p<0.01

One-tailed Mann-Whitney U test U=19 p=0.033

DAMGO-infusion in the NAc and in the VP

Sample size vehicle-treated animals n=10 Sample size NAc-infused DAMGO-treated animals n=8 Sample size VP-infused DAMGO-treated animals n=4

Mean ± SEM vehicle-treated animals 4.44±0.084 Mean ± SEM NAc-infused DAMGO-treated animals 4.525±0.077 Mean ± SEM VP-infused DAMGO-treated animals 4.44±0.204

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Shapiro-Wilk Test of Normality NAc-infused DAMGO-treated animals W=0.821 p=0.048 Shapiro-Wilk Test of Normality VP-infused DAMGO-treated animals W=0.945 p>0.05 Bartlett Test for Homogeneity of Variance K2_(2)=1.834 _p>0.05

One-way ANOVA F(2,18)=0.333 p>0.05

Two-way RM ANOVA F(10,95)=4.237 p<0.0001

Geisser-Greenhouse’s epsilon epsilon=0.550 Tukey’s multiple comparisons test

t = 0 veh-NAc veh-VP NAc-VP p>0.05 p>0.05 p>0.05 t = 5 veh-NAc veh-VP NAc-VP p>0.05 p>0.05 p>0.05 t = 10 veh-NAc veh-VP NAc-VP p>0.05 p>0.05 p>0.05 t = 20 veh-NAc veh-VP NAc-VP p>0.05 p>0.05 p>0.05 t = 30 veh-NAc veh-VP NAc-VP p>0.05 p>0.05 p>0.05 t = 60 veh-NAc veh-VP NAc-VP p>0.05 p>0.05 p>0.05

Mean ± SEM vehicle-treated animals 13.962±2.429 Mean ± SEM NAc-infused DAMGO-treated animals 12.039±2.114 Mean ± SEM VP-infused DAMGO-treated animals 14.026±5.393

Shapiro-Wilk Test of Normality vehicle-treated animals W=0.960 p>0.05 Shapiro-Wilk Test of Normality NAc-infused DAMGO-treated animals W=0.878 p>0.05 Shapiro-Wilk Test of Normality VP-infused DAMGO-treated animals W=0.871 p>0.05 Bartlett Test for Homogeneity of Variance K2_(2)=1.494 _p>0.05

One-way ANOVA F(2,19)=0.161 p>0.05

Median ± IQR vehicle-treated animals 4.6±9.77 Median ± IQR NAc-infused DAMGO-treated animals 17.25±21.923 Median ± IQR VP-infused DAMGO-treated animals 32.665±37.756

Shapiro-Wilk Test of Normality vehicle-treated animals W=0.788 p=0.007 Shapiro-Wilk Test of Normality NAc-infused

DAMGO-treated animals

W=0.921 p>0.05 Shapiro-Wilk Test of Normality VP-infused

DAMGO-treated animals

W=0.955 p>0.05 Bartlett Test for Homogeneity of Variance K2_(2)=14.039 _p<0.001

Kruskal-Wallis Test H=7.505 p=0.016

Dunn’s multiple comparisons test

veh-NAc p>0.05 veh-VP p=0.031 NAc-VP p>0.05

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Contribution of the shell in the glucoregulatory effect of DAMGO-infusion

Sample size vehicle-infused animals n=8 Sample size unilateral intra-sNAc DAMGO-infused animals n=2 Sample size bilateral intra-sNAc DAMGO-infused animals n=5

Mean ± SEM vehicle-infused animals 4.4±0.1 Mean ± SEM unilateral intra-sNAc DAMGO-infused animals 4.35±0.05 Mean ± SEM bilateral intra-sNAc DAMGO-infused animals 4.62±0.102

Shapiro-Wilk Test of Normality vehicle-infused animals W=0.908 p>0.05 Shapiro-Wilk Test of Normality unilateral intra-sNAc DAMGO-infused

animals

-

-Shapiro-Wilk Test of Normality bilateral intra-sNAc DAMGO-infused animals

W=0.884 p>0.05 Bartlett Test for Homogeneity of Variance K2_{(2)= 1.370} _p>0.05

One-way ANOVA F(2,11)=1.526 p>0.05

t = 0 veh-uni veh-bi uni-bi p>0.05 p>0.05 p>0.05 t = 5 veh-uni veh-bi uni-bi p>0.05 p>0.05 p>0.05 t = 10 veh-uni veh-bi uni-bi p>0.05 p=0.029 p>0.05 t = 20 veh-uni veh-bi p=0.007 p>0.05 uni-bi p=0.027 t = 30 veh-uni veh-bi uni-bi p=0.002 p>0.05 p>0.05 t = 60 veh-uni veh-bi uni-bi p=0.017 p>0.05 p>0.05

Shapiro-Wilk Test of Normality vehicle-infused animals W=0.980 p>0.05 Shapiro-Wilk Test of Normality unilateral intra-sNAc DAMGO-infused

animals

-

W=0.915 p>0.05 Bartlett Test for Homogeneity of Variance K2_(2)=3.661 _p>0.05

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Shapiro-Wilk Test of Normality vehicle-infused animals W=0.758 p=0.010 Shapiro-Wilk Test of Normality unilateral intra-sNAc

DAMGO-infused animals

-

W=0.8809 p>0.05 Bartlett Test for Homogeneity of Variance K2_(2)=2.972 _p>0.05

One-way ANOVA F(2,12)=6.802 p=0.011

Tukey’s multiple comparisons test

veh-uni p=0.008 veh-bi

uni-bi

p>0.05 p>0.05

Rostral-caudal effects of intra-NAc DAMGO-infusion

Sample size vehicle-infused animals n=10 Sample size rostral NAc DAMGO-infused animals n=2 Sample size caudal NAc DAMGO-infused animals n=3

Median ± IQR vehicle-infused animals 4.5±0.2 Median ± IQR rostral NAc DAMGO-infused animals 4.5±0.1 Median ± IQR caudal NAc DAMGO-infused animals 4.4±0.0

Shapiro-Wilk Test of Normality vehicle-infused animals W=0.953 p>0.05 Shapiro-Wilk Test of Normality rostral NAc DAMGO-infused animals - -Shapiro-Wilk Test of Normality caudal NAc DAMGO-infused animals - -Bartlett Test for Homogeneity of Variance K2_(2)=Inf _p<0.001

Kruskal Wallis test H=0.791 p>0.05

t = 0 veh-ros veh-cau ros-cau p>0.05 p>0.05 p>0.05 t = 5 veh-ros veh-cau ros-cau p>0.05 p>0.05 p>0.05 t = 10 veh-ros veh-cau ros-cau p>0.05 p>0.05 p>0.05 t = 20 veh-ros veh-cau ros-cau p>0.05 p>0.05 p>0.05 t = 30 veh-ros veh-cau ros-cau p>0.05 p>0.05 p>0.05 t = 60 veh-ros veh-cau ros-cau p>0.05 p>0.05 p>0.05

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Mean ± SEM vehicle-infused animals 13.962±2.429 Mean ± SEM rostral NAc DAMGO-infused animals 14.5±1.5 Mean ± SEM caudal NAc DAMGO-infused animals 10.896±4.478

Shapiro-Wilk Test of Normality vehicle-infused animals W=0.960 p>0.05 Shapiro-Wilk Test of Normality rostral NAc DAMGO-infused animals - -Shapiro-Wilk Test of Normality caudal NAc DAMGO-infused animals W=0.868 p>0.05 Bartlett Test for Homogeneity of Variance K2_(2)=1.288 _p>0.05

One-way ANOVA F(2,12)=0.223 p>0.05

Median ± IQR vehicle-infused animals 3.234±6.025 Median ± IQR rostral NAc DAMGO-infused animals 7.125±5.625 Median ± IQR caudal NAc DAMGO-infused animals 27.3±14.57

Shapiro-Wilk Test of Normality vehicle-infused animals W=0.812 p=0.021 Shapiro-Wilk Test of Normality rostral NAc DAMGO-infused animals - -Shapiro-Wilk Test of Normality caudal NAc DAMGO-infused animals W=0.919 p>0.05 Bartlett Test for Homogeneity of Variance K2_{(2)= 3.915} _p>0.05

Kruskal-Wallis Test H=4.324 p>0.05

Table S1. Details statistical analyses. Abbreviations: veh = vehicle-infused animals, NAc = NAc-infused

DAMGO-treated animals, VP = VP-infused DAMGO-DAMGO-treated animals, uni = unilateral intra-sNAc DAMGO-infused animals, bi = bilateral intra-sNAc DAMGO-infused animals, ros = rostral NAc DAMGO-infused animals, cau = caudal NAc DAMGO-infused animals.

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S2. Examination of potential effects of intra-NAc variations in cannula

placement on DAMGO-induced alteration of glycemic response to insulin

Figure S1. DAMGO-influences on insulin sensitivity may manifest in the shell of the NAc. No difference in DAMGO-influence on insulin sensitivity and CRR have been reported over the rostral-caudal axis of the NAc.

(A) Bar plot of basal glycemia of the groups, measured prior to the ivITT (t=-5). (B) Treatment effect of unilateral and bilateral DAMGO-infusion in the shell of the NAc on glycemia following an intra-arterial injection of insulin (at t=0) over time. Note that all animals were bilaterally infused; animals were assigned to groups based on infusate (vehicle or DAMGO) and whether one or two of the cannula were considered to be placed in the shell of the NAc. Values are represented as mean ± SEM. (C) Bar graph of the Negative Peak Areas of the ivITT-graphs in figure S1a. Values are represented as mean ± SEM. (D) Box plots of the area’s under the curve of the ivITT-graphs in figure S1a. Box displays the median and interquartile range, whiskers show minimum and maximum. (E) Scatter plot of basal glycemia of the groups, measured prior to the ivITT (t=-5). (F) Effects of bilateral DAMGO-infusion in the more rostral part of the NAc and the more caudal part of the NAc on glycemia following an intra-arterial injection of insulin (at t=0) over time. Error bars indicative of standard errors of the mean. (G) Bar graph of the Negative Peak Areas of the ivITT-graphs in figure S1d. Error bars indicative of standard errors of the mean. (H) Scatter plot of the Positive Peak Areas of the ivITT-graphs in figure S1d. Box displays the median and interquartile range, whiskers show minimum and maximum. *p<0.05, **p<0.01, ns = non-significant

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S3. Preliminary

data on pharmacodynamics of intra-NAc DAMGO-infusion

In the period prior to my internship, the la Fleur research group studied the pharmacodynamics of intra-NAc DAMGO-infusion. Two studies, using separate cohorts of male Wistar rats, were performed to test effects of intra-NAc DAMGO-infusion on basal glycemia (without insulin injection) and on the glycemic response to insulin after 20 minutes.

Two-way RM ANOVA (with Geisser-Greenhouse correction) was insignificant, both for treatment effects on basal glycemia (F(5,60)=0.896, p-value=0.490) and on the glycemic response to insulin after 20 minutes infusion (F(5,55)=0.225, p-value=0.950).

Figure S2. Unpublished data of the La Fleur research group showing that DAMGO-infusion in the NAc does not affect basal glycemia and has no effects on glycemic response to insulin-induced hypoglycemia after 20 minutes in male Wistar rats. (A) Treatment effect of bilateral intra-NAc DAMGO-infusion on basal glycemia over

time. Values are represented as mean ± SEM. (B) Treatment effect of DAMGO on glycemia over time, when bilaterally infused into the NAc 20 minutes before an intra-arterial injection of insulin (at t=0). Values are represented as mean ± SEM.