Dopamine D2 receptor agonist, bromocriptine, remodels adipose tissue dopaminergic signalling and upregulates catabolic pathways, improving metabolic profile in type 2 diabetes.

BACKGROUND AND OBJECTIVES: The therapeutic effects of the dopamine D2 receptor (D2R) agonist, bromocriptine, in type 2 diabetes (T2D) have been attributed to central nervous system actions. However, peripheral dopamine directly modulates glucose uptake in insulin-sensitive tissues and lipid metabolism in adipose tissue (AT). We hypothesized that the dopaminergic system may be impaired in the adipose tissue of patients with T2D and that the therapeutic actions of bromocriptine could involve the modulation of metabolism in this tissue. METHODS: The expression of dopamine receptors was evaluated in visceral AT samples from patients with obesity and stratified in several groups: insulin sensitive (IS); insulin resistance (IR) normoglycaemic; insulin resistant prediabetic; insulin resistant diabetic, according to Ox-HOMA2IR, fasting glycaemia and HbA1c levels. T2D Goto-Kakizaki rats (GK) were fed a high-caloric diet (HCD) for five months and treated with bromocriptine (10 mg/kg/day, i.p.) in the last month. The levels of dopaminergic system mediators and markers of insulin sensitivity and glucose and lipid metabolism were assessed in the peri-epididymal adipose tissue (pEWAT) and brown (BAT) adipose tissues, liver, and skeletal muscle. RESULTS: Patients with IR presented a decreasing trend of DRD1 expression in the visceral adipose tissue, being correlated with the expression of UCP1, PPARA, and insulin receptor (INSR) independently of insulin resistance and body mass index. Although no differences were observed in DRD2, DRD4 expression was significantly decreased in patients with prediabetes and T2D. In HCD-fed diabetic rats, bromocriptine increased D1R and tyrosine hydroxylase (TH) levels in pEWAT and the liver. Besides reducing adiposity, bromocriptine restored GLUT4 and PPARγ levels in pEWAT, as well as postprandial InsR activation and postabsorptive activation of lipid oxidation pathways. A reduction of liver fat, GLUT2 levels and postprandial InsR and AMPK activation in the liver was observed. Increased insulin sensitivity and GLUT4 levels in BAT and an improvement of the overall metabolic status were observed. CONCLUSIONS: Bromocriptine treatment remodels adipose tissue and the liver dopaminergic system, with increased D1R and TH levels, resulting in higher insulin sensitivity and catabolic function. Such effects may be involved in bromocriptine therapeutic effects, given the impaired expression of dopamine receptors in the visceral adipose tissue of IR patients, as well as the correlation of D1R expression with InsR and metabolic mediators.

Insulin resistance is associated with tissue-specific regulation of HIF-1α and HIF-2α during mild chronic intermittent hypoxia.

Chronic intermittent hypoxia (CIH) is a feature of obstructive sleep apnea (OSA). Whereas clinical studies have demonstrated the association between OSA and insulin resistance, the molecular mechanisms behind it are still unknown. Herein we investigated the effect of mild CIH on insulin sensitivity and we evaluated the changes in insulin and HIF signaling pathways that occur in CIH-induced insulin resistance. We showed that mild CIH obtained by 5/6 hypoxic (5%O2) cycles/h, 10.5h/day during 28 and 35 days increased arterial blood pressure. Insulin resistance and insulinemia increased with CIH duration, being significantly different after 35 days of CIH. Thirty-five days of CIH decreased insulin receptor expression and phosphorylation in skeletal muscle and adipose tissue, but not in the liver. Conversely, Glut2 expression increased in the liver of CIH-animals. Thirty-five days of CIH up-regulated HIF-1α in the liver and down-regulated HIF-1α and HIF-2α in skeletal muscle. We concluded that the effect of CIH on insulin sensitivity and signaling is time-dependent and is associated with changes in HIF signaling in insulin-sensitive tissues.

Age protects from harmful effects produced by chronic intermittent hypoxia.

Obstructive sleep apnoea (OSA) affects an estimated 3­7% of the adult population, the frequency doubling at ages >60­65 years. As it evolves, OSA becomes frequently associated with cardiovascular, metabolic and neuropsychiatric pathologies defining OSA syndrome (OSAS). Exposing experimental animals to chronic intermittent hypoxia (CIH) can be used as a model of the recurrent hypoxic and O2 desaturation patterns observed in OSA patients. CIH is an important OSA event triggering associated pathologies; CIH induces carotid body (CB)-driven exaggerated sympathetic tone and overproduction of reactive oxygen species, related to the pathogenic mechanisms of associated pathologies observed in OSAS. Aiming to discover why OSAS is clinically less conspicuous in aged patients, the present study compares CIH effects in young (3­4 months) and aged (22­24 months) rats. To define potential distinctive patterns of these pathogenic mechanisms, mean arterial blood pressure as the final CIH outcome was measured. In young rats, CIH augmented CB sensory responses to hypoxia, decreased hypoxic ventilation and augmented sympathetic activity (plasma catecholamine levels and renal artery content and synthesis rate). An increased brainstem integration of CB sensory input as a trigger of sympathetic activity is suggested. CIH also caused an oxidative status decreasing aconitase/fumarase ratio and superoxide dismutase activity. In aged animals, CIH minimally affected CB responses, ventilation and sympathetic-related parameters leaving redox status unaltered. In young animals, CIH caused hypertension and in aged animals, whose baseline blood pressure was augmented, CIH did not augment it further. Plausible mechanisms of the differences and potential significance of these findings for the diagnosis and therapy of OSAS are discussed.

Adenosine Receptor Blockade by Caffeine Inhibits Carotid Sinus Nerve Chemosensory Activity in Chronic Intermittent Hypoxic Animals.

Adenosine is a key excitatory neurotransmitter at the synapse between O(2)-sensing chemoreceptor cells-carotid sinus nerve (CSN) endings in the carotid body (CB). Herein, we have investigated the significance of adenosine, through the blockade of its receptors with caffeine, on the CB hypoxic sensitization induced by chronic intermittent hypoxia (CIH) in the rat. CIH animals were obtained by submitting rats during 15 days from 8:00 to 16:00 to 10 %O(2) for 40 s and 20 % O(2) for 80 s (i.e., 30 episodes/h). Caffeine (1 mM) was tested in spontaneous and 5 %O(2) evoked-CSN chemosensory activity in normoxic and CIH animals. CIH decreased basal spontaneous activity but increased significantly CSN activity evoked by acute hypoxia. Caffeine did not modify basal spontaneous activity in normoxic rats, but decreased significantly by 47.83 % basal activity in CIH animals. In addition, acute application of caffeine decreased 49.31 % and 56.01 % the acute hypoxic response in normoxic and CIH animals, respectively. We demonstrate that adenosine contributes to fix CSN basal activity during CIH, being also involved in hypoxic CB chemotransduction. It is concluded that adenosine participates in CB sensitization during CIH.

Hyperbaric Oxygen Therapy Improves Glucose Homeostasis in Type 2 Diabetes Patients: A Likely Involvement of the Carotid Bodies.

The carotid bodies (CBs) are peripheral chemoreceptors that respond to hypoxia increasing minute ventilation and activating the sympathetic nervous system. Besides its role in ventilation we recently described that CB regulate peripheral insulin sensitivity. Knowing that the CB is functionally blocked by hyperoxia and that hyperbaric oxygen therapy (HBOT) improves fasting blood glucose in diabetes patients, we have investigated the effect of HBOT on glucose tolerance in type 2 diabetes patients. Volunteers with indication for HBOT were recruited at the Subaquatic and Hyperbaric Medicine Center of Portuguese Navy and divided into two groups: type 2 diabetes patients and controls. Groups were submitted to 20 sessions of HBOT. OGTT were done before the first and after the last HBOT session. Sixteen diabetic patients and 16 control individual were included. Fasting glycemia was143.5 ± 12.62 mg/dl in diabetic patients and 92.06 ± 2.99 mg/dl in controls. In diabetic patients glycemia post-OGTT was 280.25 ± 22.29 mg/dl before the first HBOT session. After 20 sessions, fasting and 2 h post-OGTT glycemia decreased significantly. In control group HBOT did not modify fasting glycemia and post-OGTT glycemia. Our results showed that HBOT ameliorates glucose tolerance in diabetic patients and suggest that HBOT could be used as a therapeutic intervention for type 2 diabetes.

The Carotid Body Does Not Mediate the Acute Ventilatory Effects of Leptin.

Leptin is a hormone produced mostly in adipose tissue and playing a key role in the control of feeding and energy expenditure aiming to maintain a balance between food intake and metabolic activity. In recent years, it has been described that leptin might also contributes to control ventilation as the administration of the hormone reverses the hypoxia and hypercapnia commonly encountered in ob/ob mice which show absence of the functional hormone. In addition, it has been shown that the carotid body (CB) of the rat expresses leptin as well as the functional leptin-B receptor. Therefore, the possibility exists that the ventilatory effects of leptin are mediated by the CB chemoreceptors. In the experiments described below we confirm the stimulatory effect of leptin on ventilation, finding additionally that the CB does not mediate the instant to instant control of ventilation.

Hypoxic intensity: a determinant for the contribution of ATP and adenosine to the genesis of carotid body chemosensory activity.

Excitatory effects of adenosine and ATP on carotid body (CB) chemoreception have been previously described. Our hypothesis is that both ATP and adenosine are the key neurotransmitters responsible for the hypoxic chemotransmission in the CB sensory synapse, their relative contribution depending on the intensity of hypoxic challenge. To test this hypothesis we measured carotid sinus nerve (CSN) activity in response to moderate and intense hypoxic stimuli (7 and 0% O(2)) in the absence and in the presence of adenosine and ATP receptor antagonists. Additionally, we quantified the release of adenosine and ATP in normoxia (21% O(2)) and in response to hypoxias of different intensities (10, 5, and 2% O(2)) to study the release pathways. We found that ZM241385, an A(2) antagonist, decreased the CSN discharges evoked by 0 and 7% O(2) by 30.8 and 72.5%, respectively. Suramin, a P(2)X antagonist, decreased the CSN discharges evoked by 0 and 7% O(2) by 64.3 and 17.1%, respectively. Simultaneous application of both antagonists strongly inhibited CSN discharges elicited by both hypoxic intensities. ATP release by CB increased in parallel to hypoxia intensity while adenosine release increased preferably in response to mild hypoxia. We have also found that the lower the O(2) levels are, the higher is the percentage of adenosine produced from extracellular catabolism of ATP. Our results demonstrate that ATP and adenosine are key neurotransmitters involved in hypoxic CB chemotransduction, with a more relevant contribution of adenosine during mild hypoxia, while vesicular ATP release constitutes the preferential origin of extracellular adenosine in high-intensity hypoxia.

Carotid body function and ventilatory responses in intermittent hypoxia. Evidence for anomalous brainstem integration of arterial chemoreceptor input.

Obstructive sleep apnea is a frequent medical condition consisting in repetitive sleep-related episodes of upper airways obstruction and concurrent events of arterial blood hypoxia. There is a frequent association of cardiovascular diseases and other pathologies to this condition conforming the obstructive sleep apnea syndrome (OSAS). Laboratory models of OSAS consist in animals exposed to repetitive episodes of intermittent hypoxia (IH) which also develop cardiovascular pathologies, mostly hypertension. The overall OSAS pathophysiology appears to be linked to the repetitive hypoxia, which would cause a sensitization of carotid body (CB) chemoreflex and chemoreflex-driven hyperreactivity of the sympathetic nervous system. However, this proposal is uncertain because hyperventilation, reflecting the CB sensitization, and increased plasma CA levels, reflecting sympathetic hyperreactivity, are not constant findings in patients with OSAS and IH animals. Aiming to solve these uncertainties we have studied the entire CB chemoreflex arch in a rat model of IH, including activity of chemoreceptor cells and CB generated afferent activity to brainstem. The efferent activity was measured as ventilation in normoxia, hypoxia, and hypercapnia. Norepinephrine turnover in renal artery sympathetic endings was also assessed. Findings indicate a sensitization of the CB function to hypoxia evidenced by exaggerated chemoreceptor cell and CB afferent activity. Yet, IH rats exhibited marked hypoventilation in all studied conditions and increased turnover of norepinephrine in sympathetic endings. We conclude that IH produces a bias in the integration of the input arising from the CB with a diminished drive of ventilation and an exaggerated activation of brainstem sympathetic neurons.

A revisit to O2 sensing and transduction in the carotid body chemoreceptors in the context of reactive oxygen species biology.

Oxygen-sensing and transduction in purposeful responses in cells and organisms is of great physiological and medical interest. All animals, including humans, encounter in their lifespan many situations in which oxygen availability might be insufficient, whether acutely or chronically, physiologically or pathologically. Therefore to trace at the molecular level the sequence of events or steps connecting the oxygen deficit with the cell responses is of interest in itself as an achievement of science. In addition, it is also of great medical interest as such knowledge might facilitate the therapeutical approach to patients and to design strategies to minimize hypoxic damage. In our article we define the concepts of sensors and transducers, the steps of the hypoxic transduction cascade in the carotid body chemoreceptor cells and also discuss current models of oxygen- sensing (bioenergetic, biosynthetic and conformational) with their supportive and unsupportive data from updated literature. We envision oxygen-sensing in carotid body chemoreceptor cells as a process initiated at the level of plasma membrane and performed by a hemoprotein, which might be NOX4 or a hemoprotein not yet chemically identified. Upon oxygen-desaturation, the sensor would experience conformational changes allosterically transmitted to oxygen regulated K+ channels, the initial effectors in the transduction cascade. A decrease in their opening probability would produce cell depolarization, activation of voltage dependent calcium channels and release of neurotransmitters. Neurotransmitters would activate the nerve endings of the carotid body sensory nerve to convey the information of the hypoxic situation to the central nervous system that would command ventilation to fight hypoxia.

Adenosine in peripheral chemoreception: new insights into a historically overlooked molecule--invited article.

In the present article we review in a concise manner the literature on the general biology of adenosine signalling. In the first section we describe briefly the historical aspects of adenosine research. In the second section is presented the biochemical characteristics of this nucleoside, namely its metabolism and regulation, and its physiological actions. In the third section we have succinctly described the role of adenosine and its metabolism in hypoxia. The final section is devoted to the role of adenosine in chemoreception in the carotid body, providing a review of the literature on the presence of adenosine receptors in the carotid body; on the effects of adenosine at presynaptic level in carotid body chemoreceptor cells, as well as, its metabolism and regulation; and at postsynaptic level in carotid sinus nerve activity. Additionally, a review on the effects of adenosine in ventilation was done. This review discusses evidence for a key role of adenosine in the hypoxic response of carotid body and emphasizes new research likely to be important in the future.

The A(2B)-D(2) receptor interaction that controls carotid body catecholamines release locates between the last two steps of hypoxic transduction cascade.

We have recently demonstrated that adenosine controls the release of catecholamines (CA) from carotid body (CB) acting on A(2B) receptors. Here, we have investigated the hypothesis that this control is exerted via an interaction between adenosine A(2B) and dopamine D(2) receptors present in chemoreceptor cells and if it is, the location of this interaction on the CB hypoxic transduction cascade. Experiments were performed in vitro in CB from 3 months rats. The effect of adenosine A(2B) and dopamine D(2) receptor agonists applied alone or conjunctly, was studied on the basal and evoked release (10% O(2) and ionomycin) of CA from CB. We have observed that the inhibitory action of propylnorapomorphine, a D(2) selective agonist, on the normoxic and 10%O(2)-evoked release of CA was abolished by NECA, an A(2) agonist, meaning that an interaction between the D(2) and A(2B) receptors controls the release of CA from CB. Further, propylnorapomorphine inhibits the release of CA evoked by ionomycin, being this effect totally reversed by NECA. The present results provide direct pharmacological evidence that A(2B) and D(2) receptors interact to modulate the release of CA from rat CB between the steps of Ca(2+) entry and increase in intracellular free Ca(2+), and the activation of exocytosis and neurotransmitter release, of the stimulus-secretion coupling process.

Low glucose effects on rat carotid body chemoreceptor cells' secretory responses and action potential frequency in the carotid sinus nerve.

Glucose deprivation (hypoglycaemia) is counterbalanced by a neuroendocrine response in order to induce fast delivery of glucose to blood. Some central neurons can sense glucose, but nevertheless the most important glucose sensors/glycaemia regulators are located outside the brain. Some recent experimental evidence obtained in carotid body (CB) slices and isolated chemoreceptor cells in culture supports a role for the CB in glucose sensing and presumably glucose homeostasis, but this role has been questioned on the basis of a lack of effect of low glucose on the carotid sinus nerve activity. This work was performed in an attempt to clarify if low glucose is or is not a stimulus for the rat CB chemoreceptors. Using freshly isolated intact CB preparations we have monitored the release of catecholamines (CAs) and ATP from chemoreceptor cells in response to several concentrations of glucose, as indices of chemoreceptor cell sensitivity to glycaemia, and the electrical activity in the carotid sinus nerve (CSN), as an index of reflex-triggering output of the CB. We have observed that basal (20% O(2)) and hypoxia (7 and 10% O(2))-evoked release of CAs was identical in the presence of normal (5.55 mm) and low (3, 1 and 0 mm) glucose concentrations. 0 mm glucose did not activate the release of ATP from the CB, while hypoxia (5% O(2)) did. Basal and hypoxia (5% O(2))-induced CSN action potential frequency was identical with 5.55 and 1 mm glucose. Our results indicate that low glucose is not a direct stimulus for the rat carotid body chemoreceptors.

Caffeine inhibition of rat carotid body chemoreceptors is mediated by A2A and A2B adenosine receptors.

Caffeine, an unspecific antagonist of adenosine receptors, is commonly used to treat the apnea of prematurity. We have defined the effects of caffeine on the carotid body (CB) chemoreceptors, the main peripheral controllers of breathing, and identified the adenosine receptors involved. Caffeine inhibited basal (IC50, 210 microm) and low intensity (PO2 approximately 66 mm Hg/30 mm K+) stimulation-induced release of catecholamines from chemoreceptor cells in intact preparations of rat CB in vitro. Opposite to caffeine, 5'-(N-ethylcarboxamido)adenosine (NECA; an A2 agonist) augmented basal and low-intensity hypoxia-induced release. 2-p-(2-Carboxyethyl)phenethyl-amino-5'-N-ethylcaboxamido-adenosine hydrochloride (CGS21680), 2-hexynyl-NECA (HE-NECA) and SCH58621 (A2A receptors agents) neither affected catecholamine release nor altered the caffeine effects. The 8-cycle-1,3-dipropylxanthine (DPCPX; an A1/A2B antagonist) and 8-(4-{[(4-cyanophenyl)carbamoylmethyl]-oxy}phenyl)-1,3-di(n-propyl)xanthine (MRS1754; an A2B antagonist) mimicking of caffeine indicated that caffeine effects are mediated by A2B receptors. Immunocytochemical A2B receptors were located in tyrosine hydroxylase positive chemoreceptor cells. Caffeine reduced by 52% the chemosensory discharges elicited by hypoxia in the carotid sinus nerve. Inhibition had two components with pharmacological analysis indicating that A2A and A2B receptors mediate, respectively, the low (17 x 10(-9) m) and high (160 x 10(-6) m) IC50 effects. It is concluded that endogenous adenosine, via presynaptic A2B and postsynaptic A2A receptors, can exert excitatory effects on the overall output of the rat CB chemoreceptors.