Effects of 17β-Estradiol on Dopamine D2 Receptors in Thiamine-Deficient Female Rats: Consequences on Sucrose, Alcohol, Water Intakes and Body Weight

Our previous studies showed that 17β-estradiol (E2) modulated dopamine D2 receptor in regulating body weight set-point. The aim of this study was to understand whether thiamine deficiency influenced the E2 modulation on dopamine D2 receptors, using bromocriptine mesylate (BR) and sulpiride (SUL) as selective central dopamine-D2 receptors agonist and antagonist respectively. We studied the E2-dopamine D2 receptors interferences in a 10-day thiamine-deficient female rats for which consumptions of water, sugar, alcohol and food were daily-recorded and their consequences on body weights assessed. Our results showed that the volume of water daily ingested doubled in thiamine-deficient female rats (OXT), while sugar and alcohol consumptions collapsed with decreased weight and food consumption. On the one hand, thiamine potentiated D2/BR activity (bromocriptine-activated D2 receptors) to induce sugar intake and inhibited the same D2/BR receptors to induce water intake. On the other hand, thiamine promoted D2/SUL receptors (sulpiride-inhibited D2 receptors) for enhanced alcohol intake, increased food consumption and weight gain. Taking together, thiamine modulated the actions of 17β-estradiol on both D2/BR and D2/SUL receptors activities.


Introduction
Thiamine is an essential cofactor for several important enzymes involved in genomic actions, the transcription and synthesis of both D2L and D2S dopamine receptors isoforms [18] [19].
Recently we reported regulatory effects of E2 on D2 receptors for homeostasis needs. Our results indicated that D2S was a specific inducer of alcohol and food intakes, and increased body weight. D2S met the slow genomic actions induced by 17β-estradiol. Conversely, D2L inhibited alcohol and food intakes, but induced specifically sugar consumption, thereby regulating blood glucose levels. D2L mediated the rapid metabolic effects of E2 [20]. Our studies indicated that 17β-estradiol acted on two types of D2 receptors showing opposite functions to equilibrate energy intake vs. expenditure for weight set point regulation [20].
Together, these observations suggest that E2, thiamine and D2 receptors would be involved in a common regulatory system related to energy intake and expenditure for weight set point regulation. However, the mechanisms underlying their interactions are now being unveiled. Understanding the interactions between thiamine and E2 on D2 receptors could contribute to elucidate the mechanisms underlying overconsumption and weight gain in obese patients. To achieve this, we study the E2-dopamine D2 receptors interferences in a 10-day thiamine-deficient female rats for which consumptions of water, sugar, alcohol and food were daily-recorded. The aim of the study was to understand how these parameters interact to control body weight when thiamine is lacking.

Materials and Methods
These experiments were undertaken for further understanding of the role of thiamine (vitamin B1) on the E2-D2 receptors regulatory system whose disorder has been showed to be involved in mechanisms triggering woman obesity [20]. Since E2 is a female hormone, the present experiments are carried out exclusively on female rats.

Animals
Nulliparous females of Wistar rats, bred in our colony, which were 12 weeks old and weighing 200 -205 grams, were used in our experiments. They were maintained under standard laboratory conditions at an ambient temperature of 30˚C ± 2˚C, with light/dark cycles of 12 hours each and relative humidity reaching 85% ± 3%. Rats were individually housed in polypropylene cages (27 × 37 × 18 cm) with the floor covered by wood shavings and fed with pellet chow diet and water ad libitum. One week prior to the onset of the tests, they were acclimated to the experimental conditions. All experiments were carried out in accordance with the National Institutes of Health guide for the care and use of Laboratory animals, and the study received institutional approbation of the experiments.

Drugs and Chemicals
Drugs and Chemicals used in these experiments were: 17β-estradiol (estradiol or Germany). DMSO was the solvent used for all drug dilution [20]. Bromocriptine mesylate (BR) and sulpiride (SUL) were used as selective agonist and antagonist respectively, targeting central dopamine-D2 receptors [21] [22].

Procedures
The use of E2, BR and SUL in the present experiments was related to our previous studies indicating that 17β-estradiol acted on two types of D2 receptors showing opposite functions to equilibrate energy intake vs. expenditure for weight set point regulation [20]. However, thiamine is the rotating wheel for body energy supply [4]. The following experiments aimed to understand how the E2-D2 receptors regulatory system behaved in vitamin B1-deficient female rats for further elucidation of the mechanisms of obesity.
Within each experimental group, drinking solutions (water, 10% sucrose and 10% alcohol) were measured daily during an experimental period of 10 days.
Food consumption and body weight were also daily measured in each cage.
Drugs injections and the various measurements of weight or food and solutions intakes were performed every day at the same hour, 17:00 pm, corresponding to the beginning of activities, as the rats were nocturnal animals. Solution intake was measured by direct reading of the volume absorbed on graduated bottles, every 24 h. Fluids intake were measured in a four-bottle preference condition.
After daily drug injection, the remaining content of the vial (one drug for a non-

Experiment 2: Thiamine Deficiency
These experiments were undertaken to show whether the interactions between 17β-estradiol and dopamine D2 receptors were disrupted by thiamine deficiency. Thirty-six (36) nulliparous Wistar female rats, bred in our colony, which were 12 weeks old and weighing 200 -205 grams, were individually housed and divided into 6 treatment groups (6 rats/treatment group). In each group, each rat received oxythiamine (OXT) treatment at a dose of 20 mg/kg sc./rat/day for 10 consecutive days, to induce thiamine deficiency in rats [27]. The treatment groups were: OXT group; OXT + E2; OXT + BR; OXT + SUL; OXT + E2 + BR and OXT + E2 + SUL, for 10 consecutive days. Drugs injections procedures and doses used were identical to experiment 1. The various measurements of weight or food and solutions (10% alcohol, 10% sucrose and tap water) intakes were performed as previously. Figure 1. Effects of thiamine deficiency on 17β-estradiol (E2), bromocriptine (BR) and sulpiride (SUL) regulating water intake. The average volume of water absorbed/rat/day is represented in control (C) and OXT-treated rats (OXT). The effects of 17β-estradiol (E2), D2 (SUL and BR) receptors and their respective interactions on water intake are compared between control and thiamin-deficient rats. The consecutive 10-days average values (ml/rat/day ± SEM) of water intake within each experimental group (N = 6 female rats for one group), were represented in control (C = 6 groups) and thiamine-deficient rats (OXT = 6 groups); non-drug-treated (DMSO vehicle), 17β-estradiol (E2), sulpiride (SUL), bromocriptine (BR), E2 + SUL or E2 + BR, designating the 6 constitutive groups of control and OXT respectively. "*" denoted a significant difference between control (C) and oxythiamine-treated (OXT) female rats subjected to the same treatments, p < 0.01; "#" significantly different from their corresponding control group C or control group OXT, non-drug-treated rats, p < 0.01; " §" denoted a significant difference between hormone-D2 receptors association effects (E2 + SUL and E2 + BR) vs individual D2 receptors stimulation effects (SUL and BR) in both control and oxythiamine-treated rats p < 0.01; " ǂ" significantly different from E2 + SUL (p < 0.01).
difference among treatment groups [F (11, 600) = 51.254, p < 0.0001], with significant changes over days [F (9, 600) = 3.503, p = 0.003] and no reliable treatment × day interactions [F (99, 600) = 0.232, p = 0.99]. During the 10-day treatment period, water intake was moderated in control (C) animals (7.78 ± 0.25 ml/rat/day). Post-hoc comparisons using Fisher's PLSD test (p = 0.05) showed that B1 vitamin-deficient animals (OXT) doubled their daily volume of water ingested (14.48 ± 0.7 ml/rat/day) compared to C [p < 0.0001], indicating that in healthy animals, thiamine exerted a tonic inhibition on water consumption. Water consumption did not vary significantly when the C + E2 group (13.93 ± 0.38 ml/rat/day) was compared to the OXT + E2 group (13.91 ± 0.67 ml/rat/day), [p = 0.97], showing that thiamine did not influence directly 17β-estradiol-activated water consumption in control rats. There was also no significant difference in water consumption [p = 0.817] when the C + SUL group (9.11 ± 0.33 ml/rat/day) was compared to the OXT + SUL group (9.28 ± 0.31). These observations indicated that thiamine did not influence directly either E2 or sulpiride to regulate water consumption in the control. However, water consumption was significantly increased in the OXT + BR group (17.11 ± 0.70 ml/rat/day), compared to the C + BR group (9 ± 0.29 ml/rat/day), [p < 0.0001], indicating that thiamine exerted a tonic inhibitory action on BR activity under
compared to the C + BR group (1.18 ± 0.05 ml/rat/day), (p < 0.0001). These observations indicate that thiamine activates both D2 receptor isoforms to promote alcohol intake.

Discussion
We evaluated the effects of thiamine (B1 vitamin) deficiency on both 17β-estradiol (E2) and dopamine D2 receptors activities. Bromocriptine mesylate (BR) and sulpiride (SUL) were used as selective agonist and antagonist respectively, targeting central dopamine-D2 receptors [21] [22] and their binding receptors were described as D2/BR and D2/SUL respectively. Interferences between E2 and dopamine D2 receptors were assessed on the consumption of water, sugar, alcohol, food and the weight of thiamine-deficient female rats (OXT). Our results showed that the volume of water ingested daily doubled in B1 vitamin-deficient female rats (OXT), compared to control female rats (C), showing that in healthy animals, thiamine exerted a tonic inhibition on water consumption. Thiamine did not directly influence E2 or sulpiride to modify water consumption in control rats. However, thiamine exerted in control rats a tonic inhibition on D2/BR receptors activities which was released by thiamine deficiency. On the contrary, E2 opposed that tonic inhibition of thiamine on D2/BR receptors. In addition, E2 activated D2/SUL receptors to increase water consumption through the synergistic action of thiamine. It therefore appears that thiamine exerts a tonic inhibition on water consumption that is released by 17β-estradiol.
Our studies also showed that in thiamine-deficient female rats, sugar and al- Smith et al. [29] reported that anorexia nervosa was accompanied by thiamine deficiency and hypothermia. Administration of pharmacological doses of thiamine in such a patient improved voluntary food intake, followed by restoration of body weight and a sharp increase in sugar consumption with hyperthermia. These authors suggested the involvement of thiamine in the thermoregulation process. Molina et al. [30] showed that thiamine deficiency resulted in an increase in blood sugar (18%), lactate (3 to 4 times) and a 30% decrease in insulin.
The main functions of pancreatic beta cells are biosynthesis and the release of insulin, the only hormone that can directly reduce blood sugar levels [31]. The pancreas maintains a high level of thiamine [32], and deficiency of this vitamin negatively affects its exocrine and endocrine functions [33]. As with all other mammalian cells, pancreatic acinar cells cannot synthesize thiamine and must obtain the vitamin from the circulation through a cell membrane transport process. According to Srinivasan et al. [34], pancreatic acinar cells obtained thiamine from the circulation through thiamine membrane transporters 1 and 2 (THTR-1 and THTR-2). Pancreatic islets isolated in thiamine-deficient rats secreted less insulin. Insulin secretion in response to glucose was also decreased during thiamine deficiency. These observations show that thiamine plays an important role in insulin secretion and thiamine deficiency leads to a decrease in insulin secretion and an increase in blood glucose. This explains why in our results thiamine deficiency exacerbated water intake, while it collapsed sugar and alcohol intakes. Permanent increase in blood glucose during thiamine deficiency promoted diabetes mellitus [35] [36]. Indeed, Bâ [37] suggested that increasing extracellular glucose concentrations activated thiamine transportation from the bloodstream into both pancreatic beta cells for insulin releasing, and diverse or-gans tissues cells increasing mitochondrial ATP synthesis [38]. This process was expected to include thiamine membrane transporter coupled with a conventional G protein [39]. Since our study suggests modulating interferences between E2 and thiamine, it is conceivable that the G protein-coupled thiamine transportation may be regulated by the 17β-estradiol rapid signaling pathway. These observations suggest that, like circulating insulin, thiamine plays a major physiological role in peripheral glucose homeostasis [37]. How thiamine and 17β-estradiol control glucose homeostasis?
Our results suggest reciprocal modulating interactions between thiamine and 17β-estradiol dependent on blood glucose levels. On the one hand, thiamine exerted a tonic inhibition on water consumption that was released by 17β-estradiol.
On the other hand, 17β-estradiol exerted a tonic inhibition on sucrose intake which was released by thiamine. Under physiological conditions, D2/BR receptors mediated both actions of E2 and thiamine leading to decreased water intake and increased sucrose consumption. Indeed, bromocriptine receptor (D2L) was reported to be a specific inducer of sugar intake [20]. In addition sucrose intake collapsed in thiamine-deficient rats, showing that thiamine activated D2/BR receptors to induce sugar intake. Thiamine therefore appears to be an important regulatory factor in hypoglycemia. In conditions of high glucose demand, i.e. hypoglycemia and hyperthermia, thiamine blocked E2 inhibition on D2/BR receptors, thereby increasing sugar consumption for the body's energy supply. Literature reported 17β-estradiol to be largely involved in glucose metabolism.
Nadal et al. [31] reported that 17β-estradiol recharged blood glucose in hypoglycemia and eliminates excess blood glucose in hyperglycemia. E2 through its rapid signaling pathway triggered via the canonic estrogen receptor β (ERβ) would initiate the synthesis and increase the expression of glucose transporter 1 (GLUT-1) sensitive to blood glucose variations [40] [41], thereby facilitate glucose transport into the cells. In addition to facilitating glucose transport, E2 promoted glycolysis through the tricarboxylic acid cycle for ATP synthesis [42].
Brinton et al. [43] reported that the increase in estrogen-induced ATP synthesis was induced by the ERβ isoform of the estrogen receptor [44] improving mitochondrial respiration [45]. Moreover, ERβ increased glucose stimulated insulin secretion [46] [47]. The secreted insulin regulated blood glucose levels by removing as much glucose as possible from the circulation to enter the cells and undergo glycolysis. However, recent findings involved another estrogen receptor coupled to the G protein (GPER e.g. the old GPR30) in the regulation of E2 on insulin secretion [48]. In accordance with these findings, Koricanac et al. [49] reported selective and inhibitory interferences between insulin and estradiol signaling pathways in the context of excessive sugar intake. Indeed, the major glucose transporter in rodent islet beta cells, the glucose transporter 2 (GLUT2) associated with glucose sensing [50], was also regulated by E2. In the nervous system, GLUT2-dependent glucose sensing controlled feeding, thermoregulation and pancreatic islet cell mass and function, as well as sympathetic and parasympathetic activities [50]. According to Bian et al. [51], 17β-estradiol regulated glu-  [52] proposed that small molecules activating GPR30 may be promising in diabetes mellitus therapy. Thiamine administration alleviated diabetes mellitus [36]. We can assume that one of the small molecules involved in the regulation of estrogen receptors coupled to G proteins GPER is thiamine. Our study suggested that one of the essential functions of 17β-estradiol on sugar metabolism may be transmitted by thiamine-modulated ERβ and/or GPER receptors to ensure blood glucose homeostasis. The focal point of that regulation may be D2/BR (long chain receptor), since thiamine opposed the inhibitory actions of 17β-estradiol on D2/BR receptors, thereby increasing sugar consumption under physiological conditions, while thiamine deficiency collapsed sugar consumption. Consequently, how thiamine lack affects food consumption and body weight? Our studies showed that thiamine deficiency reduced body weight and food consumption. Under physiological conditions, thiamine contrasted with the reducing action of 17β-estradiol on weight and food consumption. In the absence of thiamine, there was an amplification of the inhibition of 17β-estradiol on the D2/SUL and D2/BR receptors resulting in a drastic decrease in weight and food consumption. On the contrary, thiamine potentiates the inducing action of sulpiride on food consumption and weight gain. E2 activated D2/SUL receptors to induce heavy alcohol and water consumption through the synergistic action of thiamine. For instance, thiamine lack collapsed the sulpiride-induced alcohol intake. Therefore, D2/SUL receptors and thiamine were involved in the mechanisms regulating hyperphagy and overweight. Supporting that hypothesis, thiamine did not participate in the reductive activity of D2 receptors (bromocriptine) on food consumption and body weight gain, but rather prevented the synergistic action of E2 on D2/BR receptors to reduce weight and food consumption. Ultimately, one of the important roles of thiamine was to stimulate weight gain and food consumption.
In humans, administration of neuroleptic drugs, which blocked dopamine receptors, caused hyperinsulinemia, increased weight gain and glucose intolerance. Conversely, treatment with bromocriptine improved glycemic control and glucose tolerance in obese type 2 diabetic patients as well as in non diabetic obese animals and humans [53]. Indeed, literature reported constantly increase in patients weights taking antipsychotic medication and sulpiride has been noted to induce severe weight gain [54] [55]. According to Baptista et al. [56] sulpiride significantly increased body weight, fat gain and food efficiency without modifying energy expenditure. Weight gain has been associated with increases in fasting glucose and lipids [57]. Selective anatgonism of D2 receptors with amisulpiride reduced largely severe weight loss and hypophagia in anorexia nervosa patients [58]. To explain the mechanisms of sulpiride-induced overweight, Parada et al. [59] reported that intrahypothalamic injections of sulpiride elicited feeding, even in satiated rats. Sulpiride microinjection in the ventromedial hy-  [60]. In addition, SULP-induced inactivation of the cAMP/protein kinase A/cAMP-response element-binding protein signaling pathway, down-regulating insulin and growth hormone release as prelude of metabolic diseases [61]. Consequently, overweight found its expression in dysregulated D2/SUL receptors [20]. Furthermore, Bâ [37] has shown that thiamine played a major physiological role in the homeostasis of body weight programming at birth, incrementation and set point regulation in offspring and adult female rats. The present studies also added that thiamine metabolism may lead to overweight when E2 is lacking as during the woman's menopause. Therefore, D2/SUL receptors and thiamine appeared to be potential inducers of woman obesity when E2 is lacking.
The present results reporting E2-thiamine reciprocal interactions on dopamine D2 receptors must be considered as preliminary, since further studies on biochemical and molecular aspects of thiamine-regulated G protein signaling are needed. Increasing interest was accorded to food selection based on macronutrients composition as important risk factor in the etiology of obesity [62]. Our future studies will address E2's ability in directing either D2L or D2S receptor toward specific nutrient intake.