membrane-potential-dye-imaging-ventromedial-hypothalamus-neurons-from

Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with ...

Universite de Bourgogne

Glucose is known to modify electrical activity of neurons in different hypothalamic areas such as the arcuate nucleus (ARC) or the ventromedian nucleus. In these structures, it has been demonstrated that glucose-induced excitation of neurons involves ATP-sensitive K(+) (K(ATP)) channel closure. The aim of the present study was to determine whether ARC neurons were able to detect high extracellular glucose concentrations and which mechanisms were involved in this detection by using whole-cell and cell-attached patch-clamp techniques in acute mouse brain slices. An increase from 5 to 20 mmol/l glucose stimulated 19% and inhibited 9% of ARC neurons. Because of the high-glucose concentrations used, we called these neurons high-glucose-excited (HGE) and high-glucose-inhibited (HGI) neurons, respectively. Glucose-induced depolarization of HGE neurons was not abolished by tetrodotoxin treatment and was correlated with an increase of membrane conductance that reversed at approximately 20 mV. Experiments with diazoxide, pinacidil, or tolbutamide showed that K(ATP) channels were present and functional in most of the ARC neurons but were mostly closed at 5 mmol/l glucose. Moreover, HGE neurons were also present in ARC of Kir6.2 null mice. These results suggested that ARC neurons have the ability to sense higher glucose concentrations than 5 mmol/l through a new K(ATP) channel-independent mechanism.

A new ATP-sensitive K+ channel-independent mechanism is involved in glucose-excited neurons of mouse arcuate nucleus.

Brain nutrient sensing allows a fine regulation of different physiological functions, such as food intake and blood glucose, related to energy homeostasis. Glucose sensing is the most studied function and a parallel has been made between the cellular mechanisms involved in pancreatic beta cells and neurons.

Brain glucose sensing: a subtle mechanism.

The ability for the brain to sense peripheral fuel availability is mainly accomplished within the hypothalamus, which detects ongoing systemic nutrients and adjusts food intake and peripheral metabolism as needed. Here, we hypothesized that mitochondrial reactive oxygen species (ROS) could trigger sensing of nutrients within the hypothalamus. For this purpose, we induced acute hypertriglyceridemia in rats and examined the function of mitochondria in the hypothalamus. Hypertriglyceridemia led to a rapid increase in the mitochondrial respiration in the ventral hypothalamus together with a transient production of ROS. Cerebral inhibition of fatty acids-CoA mitochondrial uptake prevented the hypertriglyceridemia-stimulated ROS production, indicating that ROS derived from mitochondrial metabolism. The hypertriglyceridemia-stimulated ROS production was associated with change in the intracellular redox state without any noxious cytotoxic effects, suggesting that ROS function acutely as signaling molecules. Moreover, cerebral inhibition of hypertriglyceridemia-stimulated ROS production fully abolished the satiety related to the hypertriglyceridemia, suggesting that hypothalamic ROS production was required to restrain food intake during hypertriglyceridemia. Finally, we found that fasting disrupted the hypertriglyceridemia-stimulated ROS production, indicating that the redox mechanism of brain nutrient sensing could be modulated under physiological conditions. Altogether, these findings support the role of mitochondrial ROS as molecular actors implied in brain nutrient sensing.

Role for mitochondrial reactive oxygen species in brain lipid sensing: redox regulation of food intake.

Four types of responses to glucose changes have been described in the arcuate nucleus (ARC): excitation or inhibition by low glucose concentrations <5 mmol/l (glucose-excited and -inhibited neurons) and by high glucose concentrations >5 mmol/l (high glucose-excited and -inhibited neurons). However, the ability of the same ARC neuron to detect low and high glucose concentrations has never been investigated. Moreover, the mechanism involved in mediating glucose sensitivity in glucose-inhibited neurons and the neurotransmitter identity (neuropeptide Y [NPY] or pro-opio melanocortin [POMC]) of glucosensing neurons has remained controversial. Using patch-clamp recordings on acute mouse brain slices, successive extracellular glucose changes greater than and less than 5 mmol/l show that glucose-excited, high glucose-excited, glucose-inhibited, and high glucose-inhibited neurons are different glucosensing cell subpopulations. Glucose-inhibited neurons directly detect decreased glucose via closure of a chloride channel. Using transgenic NPY-green fluorescent protein (GFP) and POMC-GFP mice, we show that 40% of NPY neurons are glucose-inhibited neurons. In contrast, <5% of POMC neurons responded to changes in extracellular glucose >5 mmol/l. In vivo results confirm the lack of glucose sensitivity of POMC neurons. Taken together, hypo- and hyperglycemia are detected by distinct populations of glucosensing neurons, and POMC and NPY neurons are not solely responsible for ARC glucosensing.

Characterization of glucosensing neuron subpopulations in the arcuate nucleus: integration in neuropeptide Y and pro-opio melanocortin networks?

A physiological adaptation to a sugar-rich meal is achieved by increased sugar uptake to match dietary load, resulting from a rapid transient translocation of the fructose/glucose GLUT2 transporter to the brush border membrane (BBM) of enterocytes. The aim of this study was to define the contributors and physiological mechanisms controlling intestinal sugar absorption, focusing on the action of insulin and the contribution of GLUT2-mediated transport.

Insulin internalizes GLUT2 in the enterocytes of healthy but not insulin-resistant mice.

Brain nutrient sensing permits fine regulation of physiological functions such as food intake and blood glucose regulation related to energy homeostasis. The mechanism of glucose sensing is the most extensively studied, and parallels have been drawn between pancreatic beta cells and neurons. Two types of glucose-sensing neuron have been identified, namely those whose activity is directly proportional to the glucose concentration, and those whose activity is inversely proportional to the glucose concentration. It was recently demonstrated that the mechanism depends on the amplitude of change in the glucose concentration. In some cases detection is probably not ensured by neurons themselves but by astrocytes, indicating that the two cell types are coupled in some way. Glucose sensing can be modulated by other nutrients (particularly fatty acids) and also by hormones (insulin, leptin and ghrelin) and peptides (NPY). The subtle cellular and molecular mechanisms involved in glucose sensing probably explain reported discrepancies in the expression of glucose transporters, hexokinases and channels. Astrocytes might also be involved in one type of response, thus adding a new level of complexity.

[Brain glucose sensing].

Nitrates such as nitroglycerin (GTN) and nitric oxide donors such as S-nitrosothiols are clinically vasoactive through stimulation of soluble guanylyl cyclase (sGC), which produces the second messenger cGMP. Development of nitrate tolerance, after exposure to GTN for several hours, is a major drawback to a widely used cardiovascular therapy. We recently showed that exposure to nitric oxide and to S-nitrosothiols causes S-nitrosylation of sGC, which directly desensitizes sGC to stimulation by nitric oxide. We tested the hypothesis that desensitization of sGC by S-nitrosylation is a mechanism of nitrate tolerance. Our results established that vascular tolerance to nitrates can be recapitulated in vivo by S-nitrosylation through exposure to cell membrane-permeable S-nitrosothiols and that sGC is S-nitrosylated and desensitized in the tolerant, treated tissues. We next determined that (1) GTN treatment of primary aortic smooth muscle cells induces S-nitrosylation of sGC and its desensitization as a function of GTN concentration; (2) S-nitrosylation and desensitization are prevented by treatment with N-acetyl-cysteine, a precursor of glutathione, used clinically to prevent development of nitrate tolerance; and (3) S-nitrosylation and desensitization are reversed by cessation of GTN treatment. Finally, we demonstrated that in vivo development of nitrate tolerance and crosstolerance by 3-day chronic GTN treatment correlates with S-nitrosylation and desensitization of sGC in tolerant tissues. These results suggest that in vivo nitrate tolerance is mediated, in part, by desensitization of sGC through GTN-dependent S-nitrosylation.

Nitroglycerin-induced S-nitrosylation and desensitization of soluble guanylyl cyclase contribute to nitrate tolerance.

Unlike the adjustable gastric banding procedure (AGB), Roux-en-Y gastric bypass surgery (RYGBP) in humans has an intriguing effect: a rapid and substantial control of type 2 diabetes mellitus (T2DM). We performed gastric lap-band (GLB) and entero-gastro anastomosis (EGA) procedures in C57Bl6 mice that were fed a high-fat diet. The EGA procedure specifically reduced food intake and increased insulin sensitivity as measured by endogenous glucose production. Intestinal gluconeogenesis increased after the EGA procedure, but not after gastric banding. All EGA effects were abolished in GLUT-2 knockout mice and in mice with portal vein denervation. We thus provide mechanistic evidence that the beneficial effects of the EGA procedure on food intake and glucose homeostasis involve intestinal gluconeogenesis and its detection via a GLUT-2 and hepatoportal sensor pathway.

Intestinal gluconeogenesis is a key factor for early metabolic changes after gastric bypass but not after gastric lap-band in mice.

Fasting increases neuropeptide Y (NPY) expression, peptide levels, and the excitability of NPY-expressing neurons in the hypothalamic arcuate (ARC) nucleus. A subpopulation of ARC-NPY neurons ( approximately 40%) are glucose-inhibited (GI)-type glucose-sensing neurons. Hence, they depolarize in response to decreased glucose. Because fasting enhances NPY neurotransmission, we propose that during fasting, GI neurons depolarize in response to smaller decreases in glucose. This increased excitation in response to glucose decreases would increase NPY-GI neuronal excitability and enhance NPY neurotransmission. Using an in vitro hypothalamic explant system, we show that fasting enhances NPY release in response to decreased glucose concentration. By measuring relative changes in membrane potential using a membrane potential-sensitive dye, we demonstrate that during fasting, a smaller decrease in glucose depolarizes NPY-GI neurons. Furthermore, incubation in low (0.7 mM) glucose enhanced while leptin (10 nM) blocked depolarization of GI neurons in response to decreased glucose. Fasting, leptin, and glucose-induced changes in NPY-GI neuron glucose sensing were mediated by 5'-AMP-activated protein kinase (AMPK). We conclude that during energy sufficiency, leptin reduces the ability of NPY-GI neurons to sense decreased glucose. However, after a fast, decreased leptin and glucose activate AMPK in NPY-GI neurons. As a result, NPY-GI neurons become depolarized in response to smaller glucose fluctuations. Increased excitation of NPY-GI neurons enhances NPY release. NPY, in turn, shifts energy homeostasis toward increased food intake and decreased energy expenditure to restore energy balance.

Fasting enhances the response of arcuate neuropeptide Y-glucose-inhibited neurons to decreased extracellular glucose.

Deregulation of hypothalamic fatty acid sensing lead to hepatic insulin-resistance which may partly contribute to further impairment of glucose homeostasis.

Deregulation of hepatic insulin sensitivity induced by central lipid infusion in rats is mediated by nitric oxide.

The response of ventromedial hypothalamic (VMH) glucose-inhibited neurons to decreased glucose is impaired under conditions where the counterregulatory response (CRR) to hypoglycemia is impaired (e.g., recurrent hypoglycemia). This suggests a role for glucose-inhibited neurons in the CRR. We recently showed that decreased glucose increases nitric oxide (NO) production in cultured VMH glucose-inhibited neurons. These in vitro data led us to hypothesize that NO release from VMH glucose-inhibited neurons is critical for the CRR.

Ventromedial hypothalamic nitric oxide production is necessary for hypoglycemia detection and counterregulation.

Hypoglycemia is the main complication for patients with type 1 diabetes mellitus receiving intensive insulin therapy. In addition to the obvious deleterious effects of acute hypoglycemia on brain function, recurrent episodes of hypoglycemia (RH) have an even more insidious effect. RH impairs the ability of the brain to detect and initiate an appropriate counterregulatory response (CRR) to restore euglycemia in response to subsequent hypoglycemia. Knowledge of mechanisms involved in hypoglycemia detection and counterregulation has significantly improved over the past 20 years. Glucose sensitive neurons (GSNs) in the ventromedial hypothalamus (VMH) may play a key role in the CRR. VMH nitric oxide (NO) production has recently been shown to be critical for both the CRR and glucose sensing by glucose-inhibited neurons. Interestingly, downstream effects of NO may also contribute to the impaired CRR after RH. In this review, we will discuss current literature regarding the molecular mechanisms by which VMH GSNs sense glucose. Putative roles of GSNs in the detection and initiation of the CRR will then be described. Finally, hypothetical mechanisms by which VMH NO production may both facilitate and subsequently impair the CRR will be discussed.

Hypothalamic nitric oxide in hypoglycemia detection and counterregulation: a two-edged sword.

Hypothalamic mitochondrial reactive oxygen species (mROS)-mediated signaling has been recently shown to be involved in the regulation of energy homeostasis. However, the upstream signals that control this mechanism have not yet been determined. Here, we hypothesize that glucose-induced mitochondrial fission plays a significant role in mROS-dependent hypothalamic glucose sensing.

Importance of mitochondrial dynamin-related protein 1 in hypothalamic glucose sensitivity in rats.

Nitric oxide (NO) by activating soluble guanylyl cyclase (sGC) is involved in vascular homeostasis via induction of smooth muscle relaxation. In cardiovascular diseases (CVDs), endothelial dysfunction with altered vascular reactivity is mostly attributed to decreased NO bioavailability via oxidative stress. However, in several studies, relaxation to NO is only partially restored by exogenous NO donors, suggesting sGC impairment. Conflicting results have been reported regarding the nature of this impairment, ranging from decreased expression of one or both subunits of sGC to heme oxidation. We showed that sGC activity is impaired by thiol S-nitrosation. Recently, angiotensin II (ANG II) chronic treatment, which induces hypertension, was shown to generate nitrosative stress in addition to oxidative stress. We hypothesized that S-nitrosation of sGC occurs in ANG II-induced hypertension, thereby leading to desensitization of sGC to NO hence vascular dysfunction. As expected, ANG II infusion increases blood pressure, aorta remodeling, and protein S-nitrosation. Intravital microscopy indicated that cremaster arterioles are resistant to NO-induced vasodilation in vivo in anesthetized ANG II-treated rats. Concomitantly, NO-induced cGMP production decreases, which correlated with S-nitrosation of sGC in hypertensive rats. This study suggests that S-nitrosation of sGC by ANG II contributes to vascular dysfunction. This was confirmed in vitro by using A7r5 smooth muscle cells infected with adenoviruses expressing sGC or cysteine mutants: ANG II decreases NO-stimulated activity in the wild-type but not in one mutant, C516A. This result indicates that cysteine 516 of sGC mediates ANG II-induced desensitization to NO in cells.

Soluble guanylyl cyclase is a target of angiotensin II-induced nitrosative stress in a hypertensive rat model.

Hormones such as leptin and ghrelin can rapidly rewire hypothalamic feeding circuits when injected into rodent brains. These experimental manipulations suggest that the hypothalamus might reorganize continually in adulthood to integrate the metabolic status of the whole body. In this study, we examined whether hypothalamic plasticity occurs in naive animals according to their nutritional conditions. For this purpose, we fed mice with a short-term high-fat diet (HFD) and assessed brain remodeling through its molecular and functional signature. We found that HFD for 3 d rewired the hypothalamic arcuate nucleus, increasing the anorexigenic tone due to activated pro-opiomelanocortin (POMC) neurons. We identified the polysialic acid molecule (PSA) as a mediator of the diet-induced rewiring of arcuate POMC. Moreover, local pharmacological inhibition and genetic disruption of the PSA signaling limits the behavioral and metabolic adaptation to HFD, as treated mice failed to normalize energy intake and showed increased body weight gain after the HFD challenge. Altogether, these findings reveal the existence of physiological hypothalamic rewiring involved in the homeostatic response to dietary fat. Furthermore, defects in the hypothalamic plasticity-driven adaptive response to HFD are obesogenic and could be involved in the development of metabolic diseases.

Food intake adaptation to dietary fat involves PSA-dependent rewiring of the arcuate melanocortin system in mice.

Hypoglycemia is a severe side effect of intensive insulin therapy. Recurrent hypoglycemia (RH) impairs the counter-regulatory response (CRR) which restores euglycemia. During hypoglycemia, ventromedial hypothalamus (VMH) production of nitric oxide (NO) and activation of its receptor soluble guanylyl cyclase (sGC) are critical for the CRR. Hypoglycemia also increases brain reactive oxygen species (ROS) production. NO production in the presence of ROS causes protein S-nitrosylation. S-nitrosylation of sGC impairs its function and induces desensitization to NO. We hypothesized that during hypoglycemia, the interaction between NO and ROS increases VMH sGC S-nitrosylation levels and impairs the CRR to subsequent episodes of hypoglycemia. VMH ROS production and S-nitrosylation were quantified following three consecutive daily episodes of insulin-hypoglycemia (RH model). The CRR was evaluated in rats in response to acute insulin-induced hypoglycemia or via hypoglycemic-hyperinsulinemic clamps. Pretreatment with the anti-oxidant N-acetyl-cysteine (NAC) was used to prevent increased VMH S-nitrosylation.

Xavier Fioramonti

Center for Taste and Feeding Behavior

Universite de Bourgogne

Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

Xavier Fioramonti

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Center for Taste and Feeding Behavior

Universite de Bourgogne

Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

Center for Taste and Feeding Behavior