Effects of glucose modulation in lateral hypothalamus on motivated behavior to obtain sucrose in an operant task.

Orexin neurons in the Lateral Hypothalamus (LH) play an important role in food seeking behavior. Approximately 60 percent of LH orexin neurons are inhibited by elevated extracellular glucose. It has been shown that elevated LH glucose decreases conditioned place preference for a food associated chamber. However, it has never been shown how modulation of LH extracellular glucose effects a rat's motivation to work for food. In this experiment we used reverse microdialysis to modulate extracellular glucose levels in LH during an operant task. Results from a progressive ratio task demonstrated that 4 mM glucose perfusion significantly decreased the animal's motivation to work for sucrose pellets while not effecting the hedonic value of the pellets. In a second experiment we demonstrated that 4 mM but not 2.5 mM glucose perfusion was sufficient to significantly decrease the number of sucrose pellets earned. Finally, we showed that modulating LH extracellular glucose mid-session from 0.7 mM to 4 mM did not affect behavior. This indicates that once feeding behavior has begun the animal becomes unresponsive to changes in extracellular glucose levels in LH. Taken together these experiments indicate that LH glucose sensing neurons play an important role in motivation to initiate feeding. However, once consumption has begun it is likely that feeding is controlled by brain regions downstream of LH.

Homeostasis Meets Motivation in the Battle to Control Food Intake.

Signals of energy homeostasis interact closely with neural circuits of motivation to control food intake. An emerging hypothesis is that the transition to maladaptive feeding behavior seen in eating disorders or obesity may arise from dysregulation of these interactions. Focusing on key brain regions involved in the control of food intake (ventral tegmental area, striatum, hypothalamus, and thalamus), we describe how activity of specific cell types embedded within these regions can influence distinct components of motivated feeding behavior. We review how signals of energy homeostasis interact with these regions to influence motivated behavioral output and present evidence that experience-dependent neural adaptations in key feeding circuits may represent cellular correlates of impaired food intake control. Future research into mechanisms that restore the balance of control between signals of homeostasis and motivated feeding behavior may inspire new treatment options for eating disorders and obesity.

Metabolic regulation of lateral hypothalamic glucose-inhibited orexin neurons may influence midbrain reward neurocircuitry.

Lateral hypothalamic area (LHA) orexin neurons modulate reward-based feeding by activating ventral tegmental area (VTA) dopamine (DA) neurons. We hypothesize that signals of peripheral energy status influence reward-based feeding by modulating the glucose sensitivity of LHA orexin glucose-inhibited (GI) neurons. This hypothesis was tested using electrophysiological recordings of LHA orexin-GI neurons in brain slices from 4 to 6week old male mice whose orexin neurons express green fluorescent protein (GFP) or putative VTA-DA neurons from C57Bl/6 mice. Low glucose directly activated ~60% of LHA orexin-GFP neurons in both whole cell and cell attached recordings. Leptin indirectly reduced and ghrelin directly enhanced the activation of LHA orexin-GI neurons by glucose decreases from 2.5 to 0.1mM by 53±12% (n=16, P<0.001) and 41±24% (n=8, P<0.05), respectively. GABA or neurotensin receptor blockade prevented leptin's effect on glucose sensitivity. Fasting increased activation of LHA orexin-GI neurons by decreased glucose, as would be predicted by these hormonal effects. We also evaluated putative VTA-DA neurons in a novel horizontal slice preparation containing the LHA and VTA. Decreased glucose increased the frequency of spontaneous excitatory post-synaptic currents (sEPSCs; 125 ± 40%, n=9, P<0.05) and action potentials (n=9; P<0.05) in 45% (9/20) of VTA DA neurons. sEPSCs were completely blocked by AMPA and NMDA glutamate receptor antagonists (CNQX 20 µM, n=4; APV 20µM, n=4; respectively), demonstrating that these sEPSCs were mediated by glutamatergic transmission onto VTA DA neurons. Orexin-1 but not 2 receptor antagonism with SB334867 (10µM; n=9) and TCS-OX2-29 (2µM; n=5), respectively, blocks the effects of decreased glucose on VTA DA neurons. Thus, decreased glucose increases orexin-dependent excitatory glutamate neurotransmission onto VTA DA neurons. These data suggest that the glucose sensitivity of LHA orexin-GI neurons links metabolic state and reward-based feeding.

Reducing neuronal nitric oxide synthase (nNOS) expression in the ventromedial hypothalamus (VMH) increases body weight and blood glucose levels while decreasing physical activity in female mice.

Glucose-inhibited (GI) neurons of the ventromedial hypothalamus (VMH) depend on neuronal nitric oxide synthase (nNOS) and AMP-activated protein kinase (AMPK) for activation in low glucose. The Lopez laboratory has shown that the effects of estrogen on brown fat thermogenesis and white fat browning require inhibition of VMH AMPK. This effect of estrogen was mediated by downstream lateral hypothalamus (LH) orexin neurons 1,2 . We previously showed that estrogen inhibits activation of GI neurons in low glucose by inhibiting AMPK 3 . Thus, we hypothesized that VMH AMPK- and nNOS-dependent GI neurons project to and inhibit orexin neurons. Estrogen inhibition of AMPK in GI neurons would then disinhibit orexin neurons and stimulate brown fat thermogenesis and white fat browning, leading to decreased body weight. To test this hypothesis, we reduced VMH nNOS expression using nNOS shRNA in female mice and measured body weight, adiposity, body temperature, white and brown fat uncoupling protein (UCP1; an index of thermogenesis and browning), locomotor activity, and blood glucose levels. Surprisingly, we saw no effect of reduced VMH nNOS expression on body temperature or UCP1. Instead, body weight and adiposity increased by 30% over 2 weeks post injection of nNOS shRNA. This was associated with increased blood glucose levels and decreased locomotor activity. We also found that VMH nNOS-GI neurons project to the LH. However, stimulation of VMH-LH projections increased excitatory glutamate input onto orexin neurons. Thus, our data do not support our original hypothesis. Excitation of orexin neurons has previously been shown to increase physical activity, leading to decreased blood glucose and body weight 4 . We now hypothesize that VMH nNOS-GI neurons play a role in this latter function of orexin neurons.

The Antinarcolepsy Drug Modafinil Reverses Hypoglycemia Unawareness and Normalizes Glucose Sensing of Orexin Neurons in Male Mice.

Perifornical hypothalamus (PFH) orexin glucose-inhibited (GI) neurons that facilitate arousal have been implicated in hypoglycemia awareness. Mice lacking orexin exhibit narcolepsy, and orexin mediates the effect of the antinarcolepsy drug modafinil. Thus, hypoglycemia awareness may require a certain level of arousal for awareness of the sympathetic symptoms of hypoglycemia (e.g., tremors, anxiety). Recurrent hypoglycemia (RH) causes hypoglycemia unawareness. We hypothesize that RH impairs the glucose sensitivity of PFH orexin GI neurons and that modafinil normalizes glucose sensitivity of these neurons and restores hypoglycemia awareness after RH. Using patch-clamp recording, we found that RH enhanced glucose inhibition of PFH orexin GI neurons in male mice, thereby blunting activation of these neurons in low-glucose conditions. We then used a modified conditioned place preference behavioral test to demonstrate that modafinil reversed hypoglycemia unawareness in male mice after RH. Similarly, modafinil restored normal glucose sensitivity to PFH orexin GI neurons. We conclude that impaired glucose sensitivity of PFH orexin GI neurons plays a role in hypoglycemia unawareness and that normalizing their glucose sensitivity after RH is associated with restoration of hypoglycemia awareness. This suggests that the glucose sensitivity of PFH orexin GI neurons is a therapeutic target for preventing hypoglycemia unawareness.

Lateral hypothalamus hypocretin/orexin glucose-inhibited neurons promote food seeking after calorie restriction.

OBJECTIVE: The present study tests the hypothesis that changes in the glucose sensitivity of lateral hypothalamus (LH) hypocretin/orexin glucose-inhibited (GI) neurons following weight loss leads to glutamate plasticity on ventral tegmental area (VTA) dopamine neurons and drives food seeking behavior. METHODS: C57BL/6J mice were calorie restricted to a 15% body weight loss and maintained at that body weight for 1 week. The glucose sensitivity of LH hypocretin/orexin GI and VTA dopamine neurons was measured using whole cell patch clamp recordings in brain slices. Food seeking behavior was assessed using conditioned place preference (CPP). RESULTS: 1-week maintenance of calorie restricted 15% body weight loss reduced glucose inhibition of hypocretin/orexin GI neurons resulting in increased neuronal activation with reduced glycemia. The effect of decreased glucose on hypocretin/orexin GI neuronal activation was blocked by pertussis toxin (inhibitor of G-protein coupled receptor subunit Gαi/o) and Rp-cAMP (inhibitor of protein kinase A, PKA). This suggests that glucose sensitivity is mediated by the Gαi/o-adenylyl cyclase-cAMP-PKA signaling pathway. The excitatory effect of the hunger hormone, ghrelin, on hcrt/ox neurons was also blocked by Rp-cAMP suggesting that hormonal signals of metabolic status may converge on the glucose sensing pathway. Food restriction and weight loss increased glutamate synaptic strength (indexed by increased AMPA/NMDA receptor current ratio) on VTA dopamine neurons and the motivation to seek food (indexed by CPP). Chemogenetic inhibition of hypocretin/orexin neurons during caloric restriction and weight loss prevented these changes in glutamate plasticity and food seeking behavior. CONCLUSIONS: We hypothesize that this change in the glucose sensitivity of hypocretin/orexin GI neurons may drive, in part, food seeking behavior following caloric restriction.

Serotonergic neurons are involved in the counter-regulatory response to hypoglycemia.

OBJECTIVES: Intensive insulin therapy provides optimal glycemic control in patients with diabetes. However, intensive insulin therapy causes so-called iatrogenic hypoglycemia as a major adverse effect. The ventromedial hypothalamus (VMH) has been described as the primary brain area initiating the counter-regulatory response (CRR). Nevertheless, the VMH receives projections from other brain areas which could participate in the regulation of the CRR. In particular, studies suggest a potential role of the serotonin (5-HT) network. Thus, the objective of this study was to determine the contribution of 5-HT neurons in CRR control. METHODS: Complementary approaches have been used to test this hypothesis in quantifying the level of 5-HT in several brain areas by HPLC in response to insulin-induced hypoglycemia, measuring the electrical activity of dorsal raphe (DR) 5-HT neurons in response to insulin or decreased glucose level by patch-clamp electrophysiology; and measuring the CRR hormone glucagon as an index of the CRR to the modulation of the activity of 5-HT neurons using pharmacological or pharmacogenetic approaches. RESULTS: HPLC measurements show that the 5HIAA/5HT ratio is increased in several brain regions including the VMH in response to insulin-induced hypoglycemia. Patch-clamp electrophysiological recordings show that insulin, but not decreased glucose level, increases the firing frequency of DR 5-HT neurons in the DR. In vivo, both the pharmacological inhibition of 5-HT neurons by intraperitoneal injection of the 5-HT1A receptor agonist 8-OH-DPAT or the chemogenetic inhibition of these neurons reduce glucagon secretion, suggesting an impaired CRR. CONCLUSION: Taken together, these data highlight a new neuronal network involved in the regulation of the CRR. In particular, this study shows that DR 5-HT neurons detect iatrogenic hypoglycemia in response to the increased insulin level and may play an important role in the regulation of CRR.

Acetylcholinesterase deficiency contributes to neuromuscular junction dysfunction in type 1 diabetic neuropathy.

Diabetic neuropathy is associated with functional and morphological changes of the neuromuscular junction (NMJ) associated with muscle weakness. This study examines the effect of type 1 diabetes on NMJ function. Swiss Webster mice were made diabetic with three interdaily ip injections of streptozotocin (STZ). Mice were severely hyperglycemic within 7 days after the STZ treatment began. Whereas performance of mice on a rotating rod remained normal, the twitch tension response of the isolated extensor digitorum longus to nerve stimulation was reduced significantly at 4 wk after the onset of STZ-induced hyperglycemia. This mechanical alteration was associated with increased amplitude and prolonged duration of miniature end-plate currents (mEPCs). Prolongation of mEPCs was not due to expression of the embryonic acetylcholine receptor but to reduced muscle expression of acetylcholine esterase (AChE). Greater sensitivity of mEPC decay time to the selective butyrylcholinesterase (BChE) inhibitor PEC suggests that muscle attempts to compensate for reduced AChE levels by increasing expression of BChE. These alterations of AChE are attributed to STZ-induced hyperglycemia since similar mEPC prolongation and reduced AChE expression were found for db/db mice. The reduction of muscle end-plate AChE activity early during the onset of STZ-induced hyperglycemia may contribute to endplate pathology and subsequent muscle weakness during diabetes.

Insulin actions on hypothalamic glucose-sensing neurones.

Subsequent to the discovery of insulin 100 years ago, great strides have been made in understanding its function, especially in the brain. It is now clear that insulin is a critical regulator of the neuronal circuitry controlling energy balance and glucose homeostasis. This review focuses on the effects of insulin and diabetes on the activity and glucose sensitivity of hypothalamic glucose-sensing neurones. We highlight the role of electrophysiological data in understanding how insulin regulates glucose-sensing neurones. A brief introduction describing the benefits and limitations of the major electrophysiological techniques used to investigate glucose-sensing neurones is provided. The mechanisms by which hypothalamic neurones sense glucose are discussed with an emphasis on those glucose-sensing neurones already shown to be modulated by insulin. Next, the literature pertaining to how insulin alters the activity and glucose sensitivity of these hypothalamic glucose-sensing neurones is described. In addition, the effects of impaired insulin signalling during diabetes and the ramifications of insulin-induced hypoglycaemia on hypothalamic glucose-sensing neurones are covered. To the extent that it is known, we present hypotheses concerning the mechanisms underlying the effects of these insulin-related pathologies. To conclude, electrophysiological data from the hippocampus are evaluated aiming to provide clues regarding how insulin might influence neuronal plasticity in glucose-sensing neurones. Although much has been accomplished subsequent to the discovery of insulin, the work described in our review suggests that the regulation of central glucose sensing by this hormone is both important and understudied.

Glucose sensing neurons in the ventromedial hypothalamus.

Neurons whose activity is regulated by glucose are found in a number of brain regions. Glucose-excited (GE) neurons increase while glucose-inhibited (GI) neurons decrease their action potential frequency as interstitial brain glucose levels increase. We hypothesize that these neurons evolved to sense and respond to severe energy deficit (e.g., fasting) that threatens the brains glucose supply. During modern times, they are also important for the restoration of blood glucose levels following insulin-induced hypoglycemia. Our data suggest that impaired glucose sensing by hypothalamic glucose sensing neurons may contribute to the syndrome known as hypoglycemia-associated autonomic failure in which the mechanisms which restore euglycemia following hypoglycemia become impaired. On the other hand, increased responses of glucose sensing neurons to glucose deficit may play a role in the development of Type 2 Diabetes Mellitus and obesity. This review will discuss the mechanisms by which glucose sensing neurons sense changes in interstitial glucose and explore the roles of these specialized glucose sensors in glucose and energy homeostasis.

Ventromedial hypothalamus glucose-inhibited neurones: A role in glucose and energy homeostasis?

The ventromedial hypothalamus (VMH) plays a complex role in glucose and energy homeostasis. The VMH is necessary for the counter-regulatory response to hypoglycaemia (CRR) that increases hepatic gluconeogenesis to restore euglycaemia. On the other hand, the VMH also restrains hepatic glucose production during euglycaemia and stimulates peripheral glucose uptake. The VMH is also important for the ability of oestrogen to increase energy expenditure. This latter function is mediated by VMH modulation of the lateral/perifornical hypothalamic area (lateral/perifornical hypothalamus) orexin neurones. Activation of VMH AMP-activated protein kinase (AMPK) is necessary for the CRR. By contrast, VMH AMPK inhibition favours decreased basal glucose levels and is required for oestrogen to increase energy expenditure. Specialised VMH glucose-sensing neurones confer the ability to sense and respond to changes in blood glucose levels. Glucose-excited (GE) neurones increase and glucose-inhibited (GI) neurones decrease their activity as glucose levels rise. VMH GI neurones, in particular, appear to be important in the CRR, although a role for GE neurones cannot be discounted. AMPK mediates glucose sensing in VMH GI neurones suggesting that, although activation of these neurones is important for the CRR, it is necessary to silence them to lower basal glucose levels and enable oestrogen to increase energy expenditure. In support of this, we found that oestrogen reduces activation of VMH GI neurones in low glucose by inhibiting AMPK. In this review, we present the evidence underlying the role of the VMH in glucose and energy homeostasis. We then discuss the role of VMH glucose-sensing neurones in mediating these effects, with a strong emphasis on oestrogenic regulation of glucose sensing and how this may affect glucose and energy homeostasis.

Lateral hypothalamic orexin glucose-inhibited neurons may regulate reward-based feeding by modulating glutamate transmission in the ventral tegmental area.

Glucose inhibits ∼60% of lateral hypothalamic (LH) orexin neurons. Fasting increases the activation of LH orexin glucose-inhibited (GI) neurons in low glucose. Increases in spontaneous glutamate excitatory postsynaptic currents (sEPSCs) onto putative VTA DA neurons in low glucose are orexin dependent (Sheng et al., 2014). VTA DA neurons modulate reward-based feeding. We tested the hypothesis that increased activation of LH orexin-GI neurons in low glucose increases glutamate signaling onto VTA DA neurons and contributes to reward-based feeding in food restricted animals. N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) currents on putative VTA DA neurons were measured using whole cell voltage clamp recording in horizontal brain slices containing the LH and VTA. Decreased glucose increased the NMDA receptor current for at least one hour after returning glucose to basal levels (P < 0.05; N = 8). The increased current was blocked by an orexin 1 receptor antagonist (P < 0.05; N = 5). Low glucose caused a similar persistent enhancement of AMPA receptor currents (P < 0.05; N = 7). An overnight fast increased the AMPA/NMDA receptor current ratio, an in vivo index of glutamate plasticity, on putative VTA DA neurons. Conditioned place preference (CPP) for palatable food was measured during LH dialysis with glucose. CPP score was negatively correlated with increasing LH glucose (P < 0.05; N = 20). These data suggest that increased activation of LH orexin-GI neurons in low glucose after weight loss, leads to enhanced glutamate signaling on VTA DA neurons, increases the drive to eat rewarding food, and may contribute to weight regain.

AMP-activated protein kinase and nitric oxide regulate the glucose sensitivity of ventromedial hypothalamic glucose-inhibited neurons.

The mechanisms by which glucose regulates the activity of glucose-inhibited (GI) neurons in the ventromedial hypothalamus (VMH) are largely unknown. We have previously shown that AMP-activated protein kinase (AMPK) increases nitric oxide (NO) production in VMH GI neurons. We hypothesized that AMPK-mediated NO signaling is required for depolarization of VMH GI neurons in response to decreased glucose. In support of our hypothesis, inhibition of neuronal nitric oxide synthase (nNOS) or the NO receptor soluble guanylyl cyclase (sGC) blocked depolarization of GI neurons to decreased glucose from 2.5 to 0.7 mM or to AMPK activation. Conversely, activation of sGC or the cell-permeable analog of cGMP, 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP), enhanced the response of GI neurons to decreased glucose, suggesting that stimulation of NO-sGC-cGMP signaling by AMPK is required for glucose sensing in GI neurons. Interestingly, the AMPK inhibitor compound C completely blocked the effect of sGC activation or 8-Br-cGMP, and 8-Br-cGMP increased VMH AMPKalpha2 phosphorylation. These data suggest that NO, in turn, amplifies AMPK activation in GI neurons. Finally, inhibition of the cystic fibrosis transmembrane regulator (CFTR) Cl(-) conductance blocked depolarization of GI neurons to decreased glucose or AMPK activation, whereas decreased glucose, AMPK activation, and 8-Br-cGMP increased VMH CFTR phosphorylation. We conclude that decreased glucose triggers the following sequence of events leading to depolarization in VMH GI neurons: AMPK activation, nNOS phosphorylation, NO production, and stimulation of sGC-cGMP signaling, which amplifies AMPK activation and leads to closure of the CFTR.

Motor unit number estimate as a predictor of motor dysfunction in an animal model of type 1 diabetes.

Peripheral neuropathy is a common complication of diabetes that leads to severe morbidity. In this study, we investigated the sensitivity of motor unit number estimate (MUNE) to detect early motor axon dysfunction in streptozotocin (STZ)-treated mice. We compared the findings with in vitro changes in the morphology and electrophysiology of the neuromuscular junction. Adult Thy1-YFP and Swiss Webster mice were made diabetic following three interdaily intraperitoneal STZ injections. Splay testing and rotarod performance assessed motor activity for 6 wk. Electromyography was carried out in the same time course, and compound muscle action potential (CMAP) amplitude, latency, and MUNE were estimated. Two-electrode voltage clamp was used to calculate quantal content (QC) of evoked transmitter release. We found that an early reduction in MUNE was evident before a detectable decline of motor activity. CMAP amplitude was not altered. MUNE decrease accompanied a drop of end-plate current amplitude and QC. We also observed small axonal loss, sprouting of nerve endings, and fragmentation of acetylcholine receptor clusters at the motor end plate. Our results suggest an early remodeling of motor units through the course of diabetic neuropathy, which can be readily detected by the MUNE technique. The early detection of MUNE anomalies is significant because it suggests that molecular changes associated with pathology and leading to neurodegeneration might already be occurring at this stage. Therefore, trials of interventions to prevent motor axon dysfunction in diabetic neuropathy should be administered at early stages of the disorder.

Corticotrophin-releasing factor receptors within the ventromedial hypothalamus regulate hypoglycemia-induced hormonal counterregulation.

Recurrent episodes of hypoglycemia impair sympathoadrenal counterregulatory responses (CRRs) to a subsequent episode of hypoglycemia. For individuals with type 1 diabetes, this markedly increases (by 25-fold) the risk of severe hypoglycemia and is a major limitation to optimal insulin therapy. The mechanisms through which this maladaptive response occurs remain unknown. The corticotrophin-releasing factor (CRF) family of neuropeptides and their receptors (CRFR1 and CRFR2) play a critical role in regulating the neuroendocrine stress response. Here we show in the Sprague-Dawley rat that direct in vivo application to the ventromedial hypothalamus (VMH), a key glucose-sensing region, of urocortin I (UCN I), an endogenous CRFR2 agonist, suppressed (approximately 55-60%), whereas CRF, a predominantly CRFR1 agonist, amplified (approximately 50-70%) CRR to hypoglycemia. UCN I was shown to directly alter the glucose sensitivity of VMH glucose-sensing neurons in whole-cell current clamp recordings in brain slices. Interestingly, the suppressive effect of UCN I-mediated CRFR2 activation persisted for at least 24 hours after in vivo VMH microinjection. Our data suggest that regulation of the CRR is largely determined by the interaction between CRFR2-mediated suppression and CRFR1-mediated activation in the VMH.

Thioredoxin-1 Overexpression in the Ventromedial Nucleus of the Hypothalamus Preserves the Counterregulatory Response to Hypoglycemia During Type 1 Diabetes in Male Rats.

We previously showed that the glutathione precursor, N-acetylcysteine (NAC), prevented hypoglycemia-associated autonomic failure (HAAF) and impaired activation of ventromedial hypothalamus (VMH) glucose-inhibited (GI) neurons by low glucose after recurrent hypoglycemia (RH) in nondiabetic rats. However, NAC does not normalize glucose sensing by VMH GI neurons when RH occurs during diabetes. We hypothesized that recruiting the thioredoxin (Trx) antioxidant defense system would prevent HAAF and normalize glucose sensing after RH in diabetes. To test this hypothesis, we overexpressed Trx-1 (cytosolic form of Trx) in the VMH of rats with streptozotocin (STZ)-induced type 1 diabetes. The counterregulatory response (CRR) to hypoglycemia in vivo and the activation of VMH GI neurons in low glucose using membrane potential sensitive dye in vitro was measured before and after RH. VMH Trx-1 overexpression normalized both the CRR and glucose sensing by VMH GI neurons in STZ rats. VMH Trx-1 overexpression also lowered the insulin requirement to prevent severe hyperglycemia in STZ rats. However, like NAC, VMH Trx-1 overexpression did not prevent HAAF or normalize activation of VMH GI neurons by low glucose in STZ rats after RH. We conclude that preventing HAAF in type 1 diabetes may require the recruitment of both antioxidant systems.

Hypoglycemia: Role of Hypothalamic Glucose-Inhibited (GI) Neurons in Detection and Correction.

Hypoglycemia is a profound threat to the brain since glucose is its primary fuel. As a result, glucose sensors are widely located in the central nervous system and periphery. In this perspective we will focus on the role of hypothalamic glucose-inhibited (GI) neurons in sensing and correcting hypoglycemia. In particular, we will discuss GI neurons in the ventromedial hypothalamus (VMH) which express neuronal nitric oxide synthase (nNOS) and in the perifornical hypothalamus (PFH) which express orexin. The ability of VMH nNOS-GI neurons to depolarize in low glucose closely parallels the hormonal response to hypoglycemia which stimulates gluconeogenesis. We have found that nitric oxide (NO) production in low glucose is dependent on oxidative status. In this perspective we will discuss the potential relevance of our work showing that enhancing the glutathione antioxidant system prevents hypoglycemia associated autonomic failure (HAAF) in non-diabetic rats whereas VMH overexpression of the thioredoxin antioxidant system restores hypoglycemia counterregulation in rats with type 1 diabetes.We will also address the potential role of the orexin-GI neurons in the arousal response needed for hypoglycemia awareness which leads to behavioral correction (e.g., food intake, glucose administration). The potential relationship between the hypothalamic sensors and the neurocircuitry in the hindbrain and portal mesenteric vein which is critical for hypoglycemia correction will then be discussed.

Oleic Acid in the Ventral Tegmental Area Inhibits Feeding, Food Reward, and Dopamine Tone.

Long-chain fatty acids (FAs) act centrally to decrease food intake and hepatic glucose production and alter hypothalamic neuronal activity in a manner that depends on FA type and cellular transport proteins. However, it is not known whether FAs are sensed by ventral tegmental area (VTA) dopamine (DA) neurons to control food-motivated behavior and DA neurotransmission. We investigated the impact of the monounsaturated FA oleate in the VTA on feeding, locomotion, food reward, and DA neuronal activity and DA neuron expression of FA-handling proteins and FA uptake. A single intra-VTA injection of oleate, but not of the saturated FA palmitate, decreased food intake and increased locomotor activity. Furthermore, intra-VTA oleate blunted the rewarding effects of high-fat/sugar food in an operant task and inhibited DA neuronal firing. Using sorted DA neuron preparations from TH-eGFP mice we found that DA neurons express FA transporter and binding proteins, and are capable of intracellular transport of long-chain FA. Finally, we demonstrate that a transporter blocker attenuates FA uptake into DA neurons and blocks the effects of intra-VTA oleate to decrease food-seeking and DA neuronal activity. Together, these results suggest that DA neurons detect FA and that oleate has actions in the VTA to suppress DA neuronal activity and food seeking following cellular incorporation. These findings highlight the capacity of DA neurons to act as metabolic sensors by responding not only to hormones but also to FA nutrient signals to modulate food-directed behavior.

Glucokinase is a critical regulator of ventromedial hypothalamic neuronal glucosensing.

To test the hypothesis that glucokinase is a critical regulator of neuronal glucosensing, glucokinase activity was increased, using a glucokinase activator drug, or decreased, using RNA interference combined with calcium imaging in freshly dissociated ventromedial hypothalamic nucleus (VMN) neurons or primary ventromedial hypothalamus (VMH; VMN plus arcuate nucleus) cultures. To assess the validity of our approach, we first showed that glucose-induced (0.5-2.5 mmol/l) changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) oscillations, using fura-2 and changes in membrane potential (using a membrane potential-sensitive dye), were highly correlated in both glucose-excited and -inhibited neurons. Also, glucose-excited neurons increased (half-maximal effective concentration [EC(50)] = 0.54 mmol/l) and glucose-inhibited neurons decreased (half-maximal inhibitory concentration [IC(50)] = 1.12 mmol/l) [Ca(2+)](i) oscillations to incremental changes in glucose from 0.3 to 5 mmol/l. In untreated primary VMH neuronal cultures, the expression of glucokinase mRNA and the number of demonstrable glucosensing neurons fell spontaneously by half over 12-96 h without loss of viable neurons. Transfection of neurons with small interfering glucokinase RNA did not affect survival but did reduce glucokinase mRNA by 90% in association with loss of all demonstrable glucose-excited neurons and a 99% reduction in glucose-inhibited neurons. A pharmacological glucokinase activator produced a dose-related increase in [Ca(2+)](i) oscillations in glucose-excited neurons (EC(50) = 0.98 mmol/l) and a decrease in glucose-inhibited neurons (IC(50) = 0.025 micromol/l) held at 0.5 mmol/l glucose. Together, these data support a critical role for glucokinase in neuronal glucosensing.

Brain GLUT4 Knockout Mice Have Impaired Glucose Tolerance, Decreased Insulin Sensitivity, and Impaired Hypoglycemic Counterregulation.

GLUT4 in muscle and adipose tissue is important in maintaining glucose homeostasis. However, the role of insulin-responsive GLUT4 in the central nervous system has not been well characterized. To assess its importance, a selective knockout of brain GLUT4 (BG4KO) was generated by crossing Nestin-Cre mice with GLUT4-floxed mice. BG4KO mice had a 99% reduction in GLUT4 protein expression throughout the brain. Despite normal feeding and fasting glycemia, BG4KO mice were glucose intolerant, demonstrated hepatic insulin resistance, and had reduced glucose uptake in the brain. In response to hypoglycemia, BG4KO mice had impaired glucose sensing, noted by impaired epinephrine and glucagon responses and impaired c-fos activation in the hypothalamic paraventricular nucleus. Moreover, in vitro glucose sensing of glucose-inhibitory neurons from the ventromedial hypothalamus was impaired in BG4KO mice. In summary, BG4KO mice are glucose intolerant, insulin resistant, and have impaired glucose sensing, indicating a critical role for brain GLUT4 in sensing and responding to changes in blood glucose.