Ramón Alvarez-Buylla

Carotid Sinus Receptors Participate in Glucose Homeostasis

This paper describes (a) the influence of glucose on carotid chemoreceptor activity, and (b) the participation of carotid receptors in glucose homeostasis. After eliminating the carotid body baroreceptors in anesthetized cats, the injection of glucose to the vascularly isolated carotid sinus region reduced by 20% the electrical activity of carotid body chemoreceptors and increased their threshold to hypoxia. Mannitol in the same concentration did not change the chemoreceptor activity. A decrease in baroreceptor activity elicited by carotid occlusion, or carotid chemoreceptor stimulation with 50 micrograms/kg cyanide (NaCN), produced an immediate increase in the output of hepatic glucose, raising the hepatic venous-arterial glucose difference above basal levels. Bilateral adrenalectomy eliminated these reflex responses. Cyanide injected in the same conditions caused a sharp increase in glucose retention by the brain. In control experiments, after sectioning the carotid nerve, NaCN injections were ineffective. However, electrical stimulation of the central stump of carotid nerve elicited reflex effects similar to those obtained with NaCN stimulation.

Changes in blood glucose concentration in the carotid body-sinus modify brain glucose retention

To test whether blood glucose concentration in the carotid body-sinus may influence the amount of glucose retained by the brain, the isolated carotid sinus was perfused with glucose-rich blood or glucose-poor blood from a second animal. The circulation of the right carotid body-sinus was temporarily isolated in rat A, and perfused with blood coming from rat B. Blood glucose in rat B was modified by injections of glucose or insulin. Changes in glucose retention by the brain were measured in rat A. When the isolated carotid body-sinus in rat A was perfused with hyperglycemic blood (16.7 mM), brain glucose retention in rat A decreased significantly from 0.14 +/- 0.02 mumol/g/min (t = 0) to 0.08 +/- 0.01 mumol/g/min at 4 min after the beginning of perfusion. In contrast, the perfusion of the isolated carotid body-sinus of rat A with hypoglycemic blood (2.7 mM) from rat B, had the opposite effect. Brain glucose retention in rat A increased (0.23 +/- 0.03 mumol/g/min) at t = 4 min in comparison to control values (0.13 +/- 0.01 mumol/g/min). Chemoreceptor activity was also manipulated by the injection of cyanide (NaCN) in rat B, under these conditions, brain glucose retention in rat A increased from 0.13 +/- 0.01 mumol/g/min to 0.28 +/- 0.03 mumol/g/min between 4 to 8 min after the beginning of perfusion. These results indicate that chemosensory activity within the carotid body-sinus, superfused in vivo with different glucose concentrations, modify glucose retention by the brain.

Arginine-vasopressin in nucleus of the tractus solitarius induces hyperglycemia and brain glucose retention

Hypothalamic arginine-vasopressin (AVP) plays an important role both as a neurotransmitter and hormone in the regulation of blood glucose and feeding behavior. AVP-containing axons from the parvocellular subdivision of paraventricular nucleus of the hypothalamus terminate in the nucleus of the tractus solitarius (NTS), but the function of this projection is not known. Interestingly, the NTS also receives afferent information from the carotid body and other peripheral receptors involved in glucose homeostasis. We have previously reported that stimulation of the carotid body receptors initiates a hyperglycemic reflex and increases brain glucose retention. Here we show that direct administration of micro-doses of AVP into the NTS of anesthetized or awake rats rapidly increased the levels of blood glucose concentration and brain arterio-venous (A-V) glucose difference. This effect was not observed when the same doses of AVP were injected in the brainstem outside NTS. Arginine-vasopressin antagonist microinjections alone produced a small but significant reduction in brain A-V glucose. Pre-administered VP1-receptor antagonist [beta-mercapto-beta,beta-cyclopentamethylene-propionyl(1),O-Me-Tyr(2),Arg(8)]vasopressin blocked the effects of AVP. These results indicate that AVP acting on its receptors locally within the NTS participates in glucose homeostasis, increasing both blood glucose concentration and brain A-V glucose differences. Hypothalamic AVP may facilitate hyperglycemic responses initiated by peripheral signals processed at the level of the NTS.

GabaB receptors activation in the NTS blocks the glycemic responses induced by carotid body receptor stimulation

The carotid body receptors participate in glucose regulation sensing glucose levels in blood entering the cephalic circulation. The carotid body receptors information, is initially processed within the nucleus tractus solitarius (NTS) and elicits changes in circulating glucose and brain glucose uptake. Previous work has shown that gamma-aminobutyric acid (GABA) in NTS modulates respiratory reflexes, but the role of GABA within NTS in glucose regulation remains unknown. Here we show that GABA(B) receptor agonist (baclofen) or antagonists (phaclofen and CGP55845A) locally injected into NTS modified arterial glucose levels and brain glucose retention. Control injections outside NTS did not elicit these responses. In contrast, GABA(A) agonist and antagonist (muscimol or bicuculline) produced no significant changes in blood glucose levels. When these GABAergic drugs were applied before carotid body receptors stimulation, again, only GABA(B) agonist or antagonist significantly affected glycemic responses; baclofen microinjection significantly reduced the hyperglycemic response and brain glucose retention observed after carotid body receptors stimulation, while phaclofen produced the opposite effect, increasing significantly hyperglycemia and brain glucose retention. These results indicate that activation of GABA(B), but not GABA(A), receptors in the NTS modulates the glycemic responses after anoxic stimulation of the carotid body receptors, and suggest the presence of a tonic inhibitory mechanism in the NTS to avoid hyperglycemia.

Induction of brain glucose uptake by a factor secreted into cerebrospinal fluid

It is well established that the carotid body receptors (CBR), at the bifurcation of the carotid artery, inform the brain of changes in the concentration of CO(2) and O(2) in arterial blood. More recent work suggests that these receptors are also extremely sensitive to blood glucose levels suggesting that they may play an important role as sensors of blood components important for brain energy metabolism. Much less is known about changes in brain glucose metabolism in response to CBR activation. Here we show that 2-8 min after local injection of sodium cyanide (NaCN) into the CBR or after electrical stimulation of the carotid sinus nerve in dogs and rats, brain glucose uptake increased fourfold. Cerebrospinal fluids (CSF) transferred from dogs, 2-8 min after CBR stimulation, into the cisterna magna of non-stimulated dogs or rats induced a similar increase in brain glucose uptake. CSF from stimulated dogs was also active when injected intravenously in anesthetized or awake rats. The activity was destroyed when the stimulated CSF was heated to 100 degrees C or treated with trypsin. We conclude that a peptide important for brain glucose regulation appears in the CSF shortly after CBR stimulation.

Nitric Oxide in Brain Glucose Retention after Carotid Body Receptors Stimulation with Cyanide in Rats

In contrast to most other tissues, which exhibit considerable flexibility with respect to the nature of the substrates for their energy metabolism, the normal brain is restricted almost exclusively to glucose due to its distinguishing characteristics in vivo. Actual glucose utilization is 31 μmol/100 g tissue/min, in the normal, conscious human brain, indicating that glucose consumption is in excess for total oxygen consumption (Sokoloff, 1991). Although present in low concentration in brain (3.3 mmol/kg in rat), glycogen is a unique energy reserve for initiation of its metabolism. However, if glycogen concentration in the brain were the sole supply, normal energetic requirements would be maintained for less than 5 min (Sokoloff, 1991). While the brain contains insulin receptors, and insulin-responsive glucose transporters, the role of insulin in the regulation of brain glucose metabolism is controversial (Obici et al., 2002). The carotid body receptors (CBR) are sensitive to glucose (Alvarez-Buylla and Alvarez-Buylla, 1988, 1994, Pardal and Lopez Barneo, 2002) and play an important role in the insulin-induced counterregulatory response to mild hypoglycemia (Koyama et al., 2000). Local stimulation of CBR by cyanide (NaCN), or local low glucose levels in the isolated carotid sinus (CS), have been shown to promptly increase the activity in the carotid sinus nerve, that in turn trigger an enhancement in glucose retention by the brain (BGR) (Alvarez-Buylla et al., 1994). In contrast, this effect is not observed in animals with denervated carotid bodies (AlvarezBuylla and Alvarez-Buylla, 1988). The central mechanism that mediates the previously mentioned glycemic responses is unknown, but other studies from our laboratory suggest the participation of arginine-vasopressin (AVP), the endogenous ligand for the V1a vasopressin receptor, as the effector mediator in this response (Montero et al., 2003). AVP is widely synthesized in the brain, including the paraventricular, supraoptic and suprachiasmatic nuclei of the hypothalamus, and has been related to nitric oxide (NO) function in brain (Kadekaro et al., 1998). There are evidences that NO, an intercellular signaling

Carotid chemoreceptors participation in brain glucose regulation. Role of arginine-vasopressin.

In previous studies we suggested that carotid body receptors (CBR) participate in glucose homeostasis (Alvarez-Buylla and Alvarez-Buylla, 1994). One of the most striking effects of the carotid chemoreceptor stimulation with cyanide (NaCN) is a rapid hyperglycemic reflex with glucose retention by the brain (Alvarez-Buylla et al., 1996). Pituitary and adrenals, two glands involved in glucose homeostasis, participate in the efferent pathway of this reflex (Alvarez-Buylla, 1997). Surgical removal of the neurohypophysis but not the anterior hypophysis abolishes the hyperglycemic reflex initiated in the CBR. Some of these efferent effects may be mediated through the direct action of neurohypophysial hormones on liver and adrenals. It has become progressively apparent that, in addition to its antidiuretic and vasopressor effects, vasopressin (AVP) also displays a powerful glycogenolytic action on the liver (Hems et al., 1978; Morel et al., 1992) and modulates glucose metabolism when the organism is under stress (Wideman and Murphy, 1993). Although AVP is widely distributed throughout the central nervous system (CNS) (Ostrowski et al., 1994), and it is known to act as an excitatory transmitter (Jakab et al., 1991), the effect of AVP on cerebral glucose homeostasis has not been documented. Importantly, carotid sinus perfusion with deoxygenated blood (Share and Levy, 1966) or following bilateral carotid occlusion (Harris, 1979), results in an increase in AVP levels in plasma. Carotid body receptor

Effects of Intracisternal Glucose or Insulin Injections on Glucose Homeostasis in Cat

The injection of glucose (100 mg) into the cisterna magna of intact anesthetized cats elicited immediate glycosurie and natriuresis without significant changes in blood glucose concentration. Immunoreactive insulin (IRI) increased 140% in plasma, and Na+ concentration decreased in cerebrospinal fluid (CSF). After kidney denervation there was a significant decrease in glucose and Na+ concentrations in urine. Control injections with manitol did not elicit changes in the studied parameters. Abdominal vagotomy abolished the rise in IRI levels and the decrease in Na+ concentration in CSF. Vagotomy or adrenalectomy also attenuated the glycosurie and the rise in urine Na+ concentration. The intracisternal injection of insulin (0.5 U/kg) caused first, a decrease in glucose concentration in CSF and afterwards a longer latency in plasma. Again, these responses were significantly attenuated when insulin was administered in vagotomized cats. These experiments indicate that the nervous system, through the vagi, adrenal glands, and kidneys, plays an important role in glucose homeostasis after increasing glucose or insulin levels in the CSF above physiologic concentrations. The results obtained with a denervated kidney confirm the participation of nervous system in the effector mechanism that brings the sugar and Na+ into the urine. Evidence is presented for an interrelationship between glucose and Na+ concentrations in blood, urine, and CSF.

Enhancing Effect of Vasopressin on the Hyperglycemic Response to Carotid Body Chemoreceptor Stimulation

Glucose homeostasis, a fundamental process for life, is controlled at multiple levels. Glucose sensitive receptors in the brain, portal vein, liver, pancreas and carotid bodies (Alvarez-Buylla and Roces de Alvarez-Buylla, 1994) provide afferent information to central nervous system (CNS) about the glucose concentration in different regions of the body. In the CNS, this input is integrated by the hypothalamus and the nucleus of the tractus solitarius (NTS) (Adachi et al., 1995). Additionally, there is evidence that carotid body receptors (CBR) are also sensitive to changes in blood glucose concentration (Alvarez- Buylla and Roces de Alvarez-Buylla, 1994; Lopez-Barneo et al., 2001) and afferent impulses from these receptors induce a reflex response on glucose levels: 1) by enhancing glucose production by the liver, and 2 by promoting glucose retention by the brain. Carotid bodies play an important role in the insulin-induced counterregulatory response to mild hypoglycemia (Koyama et al., 2000). The efferent pathway for these reflexes is not fully understood, but previous experiments identify the neurohypophysis and adrenal glands as necessary for the hyperglycemic reflex initiated by NaCN stimulation, and suggest that the effects of these two glands on CBR hyperglycemic reflex are humoral (Alvarez-Buylla et al., 1997). This is supported by the finding that the neurohypophyseal hormone arginine-vasopressin (AVP) has a modulatory role on glucose metabolism during stress, and that an increase of vasopressin plasma levels is observed after perfusion of the carotid sinus with deoxygenated blood, a method similar to NaCN stimulation (Share and Levy, 1966). In addition, hypophysectomy leads to adrenal cortical atrophy and hypoglycemia (Wurtman et al., 1968). We have previously hypothesized that pituitary AVP may be involved in the hyperglycemic reflex initiated by CBR stimulation. In this paper we extend the study to the role of glucose in regulating AVP at the level of NTS (Yarkov et al., 2001), and suggest that this peptide may facilitate hyperglycemic reflexes elicited by CBR stimulation. We show that AVP can directly trigger a hyperglycemic reflex similar to that obtained after CBR stimulation. We suggest that AVP may interact with vasopressin receptors located in the NTS, liver, adrenal cells and pancreas to stimulate the secretion of epinephrine (E) and glucagon.

Functional Activation of Cerebral Glucose Uptake after Carotid Body Stimulation

Our laboratory is interested in the mechanism of glucose homeostasis (AlvarezBuylla & Roces de Alvarez-Buylla, 1975) and in particular in how appropriate levels of glucose are ensured for brain metabolism. The central nervous system (CNS) relies on a large and sustained supply of glucose for its functional activity (Erecinska & Silver, 1989; Ueki et al, 1988). However, the mechanisms that regulate the transfer of glucose from blood to brain are not well understood. Insulin increases membrane transfer of glucose in many tissues, but it does not regulate glucose uptake by the brain (LeMay et al, 1988). Besides its neuroendocrine function, the hypothalamus has an important role in integrating the activity of the autonomic nervous system (Chen et al, 1994), but its possible participation in regulating brain glucose uptake is unknown. In a previous study we have shown that changes in blood glucose concentration in the carotid body modify brain glucose retention (Alvarez-Buylla & Roces de Alvarez-Buylla, 1994). The role of the cerebrospinal fluid (CSF) in neuroendocrine functions, and as a conduit of communication between hypothalamus, pituitary and brain has been established (Jackson, 1984). In this paper we show that after carotid body receptor (CBR) stimulation a putative bioactive substance appears in the CSF that increases glucose retention in the brain. We further study the pituitary and adrenal glands participation in this reflex effect caused by CBR stimulation.