IL-6 ameliorates defective leptin sensitivity in DIO ventromedial hypothalamic nucleus neurons.

Rats selectively bred to develop diet-induced obesity (DIO) have an early onset reduction in the sensitivity of their ventromedial hypothalamic nucleus (VMN) neurons to leptin compared with diet-resistant (DR) rats. This reduced sensitivity includes decreased leptin receptor (Lepr-b) mRNA expression, leptin receptor binding, leptin-induced phosphorylation of STAT3 (pSTAT3), and impaired leptin excitation (LepE) of VMN neurons. When administered exogenously, the pancreatic peptide, amylin, acts synergistically to reduce food intake and body weight in obese, leptin-resistant DIO rats by increasing VMN leptin signaling, likely by stimulation of microglia IL-6, which acts on its receptor to increase leptin-induced pSTAT3. Here, we demonstrate that incubation of cultured VMN neurons of outbred rats with IL-6 increases their leptin sensitivity. Control, dissociated DIO VMN neurons express 66% less Lepr-b and 75% less Bardet Biedl Syndrome-6 (BBS6) mRNA and have reduced leptin-induced activation of LepE neurons compared with DR neurons. Incubation for 4 days with IL-6 increased DIO neuron Lepr-b expression by 77% and BBS6 by 290% and corrected their defective leptin activation of LepE neurons to DR levels. Since BBS6 enhances trafficking of Lepr-b to the cell membrane, the increases in Lepr-b and BBS6 expression appear to account for correction of the reduced leptin excitation of DIO LepE neurons to that of control DR rats. These data support prior findings suggesting that IL-6 mediates the leptin-sensitizing effects of amylin on VMN neurons and that the inherent leptin resistance of DIO rats can be effectively reversed at a cellular level by IL-6.

Orexin signaling is necessary for hypoglycemia-induced prevention of conditioned place preference.

While the neural control of glucoregulatory responses to insulin-induced hypoglycemia is beginning to be elucidated, brain sites responsible for behavioral responses to hypoglycemia are relatively poorly understood. To help elucidate central control mechanisms associated with hypoglycemia unawareness, we first evaluated the effect of recurrent hypoglycemia on a simple behavioral measure, the robust feeding response to hypoglycemia, in rats. First, food intake was significantly, and similarly, increased above baseline saline-induced intake (1.1 ± 0.2 g; n = 8) in rats experiencing a first (4.4 ± 0.3; n = 8) or third daily episode of recurrent insulin-induced hypoglycemia (IIH, 3.7 ± 0.3 g; n = 9; P < 0.05). Because food intake was not impaired as a result of prior IIH, we next developed an alternative animal model of hypoglycemia-induced behavioral arousal using a conditioned place preference (CPP) model. We found that hypoglycemia severely blunted previously acquired CPP in rats and that recurrent hypoglycemia prevented this blunting. Pretreatment with a brain penetrant, selective orexin receptor-1 antagonist, SB-334867A, blocked hypoglycemia-induced blunting of CPP. Recurrently hypoglycemic rats also showed decreased preproorexin expression in the perifornical hypothalamus (50%) but not in the adjacent lateral hypothalamus. Pretreatment with sertraline, previously shown to prevent hypoglycemia-associated glucoregulatory failure, did not prevent blunting of hypoglycemia-induced CPP prevention by recurrent hypoglycemia. This work describes the first behavioral model of hypoglycemia unawareness and suggests a role for orexin neurons in mediating behavioral responses to hypoglycemia.

Early postnatal amylin treatment enhances hypothalamic leptin signaling and neural development in the selectively bred diet-induced obese rat.

Selectively bred diet-induced obese (DIO) rats become obese on a high-fat diet and are leptin resistant before becoming obese. Compared with diet-resistant (DR) neonates, DIO neonates have impaired leptin-dependent arcuate (ARC) neuropeptide Y/agouti-related peptide (NPY/AgRP) and α-melanocyte-stimulating hormone (α-MSH; from proopiomelanocortin (POMC) neurons) axon outgrowth to the paraventricular nucleus (PVN). Using phosphorylation of STAT3 (pSTAT3) as a surrogate, we show that reduced DIO ARC leptin signaling develops by postnatal day 7 (P7) and is reduced within POMC but not NPY/AgRP neurons. Since amylin increases leptin signaling in adult rats, we treated DIO neonates with amylin during postnatal hypothalamic development and assessed leptin signaling, leptin-dependent ARC-PVN pathway development, and metabolic changes. DIO neonates treated with amylin from P0-6 and from P0-16 increased ARC leptin signaling and both AgRP and α-MSH ARC-PVN pathway development, but increased only POMC neuron number. Despite ARC-PVN pathway correction, P0-16 amylin-induced reductions in body weight did not persist beyond treatment cessation. Since amylin enhances adult DIO ARC signaling via an IL-6-dependent mechanism, we assessed ARC-PVN pathway competency in IL-6 knockout mice and found that the AgRP, but not the α-MSH, ARC-PVN pathway was reduced. These results suggest that both leptin and amylin are important neurotrophic factors for the postnatal development of the ARC-PVN pathway. Amylin might act as a direct neurotrophic factor in DIO rats to enhance both the number of POMC neurons and their α-MSH ARC-PVN pathway development. This suggests important and selective roles for amylin during ARC hypothalamic development.

Endogenous VMH amylin signaling is required for full leptin signaling and protection from diet-induced obesity.

Amylin enhances arcuate (ARC) and ventromedial (VMN) hypothalamic nuclei leptin signaling and synergistically reduces food intake and body weight in selectively bred diet-induced obese (DIO) rats. Since DIO (125)I-amylin dorsomedial nucleus-dorsomedial VMN binding was reduced, we postulated that this contributed to DIO ventromedial hypothalamus (VMH) leptin resistance, and that impairing VMH (ARC + VMN) calcitonin receptor (CTR)-mediated signaling by injecting adeno-associated virus (AAV) expressing a short hairpin portion of the CTR mRNA would predispose diet-resistant (DR) rats to obesity on high-fat (45%) diet (HFD). Depleting VMH CTR by 80-90% in 4-wk-old male DR rats reduced their ARC and VMN (125)I-labeled leptin binding by 57 and 51%, respectively, and VMN leptin-induced phospho-signal transducer and activator of transcription 3-positive neurons by 59% vs. AAV control rats. After 6 wk on chow, VMH CTR-depleted DR rats ate and gained the equivalent amount of food and weight but had 18% heavier fat pads (relative to carcass weight), 144% higher leptin levels, and were insulin resistant compared with control AAV DR rats. After 6 wk more on HFD, VMH CTR-depleted DR rats ate the same amount but gained 28% more weight, had 60% more carcass fat, 254% higher leptin levels, and 132% higher insulin areas under the curve during an oral glucose tolerance test than control DR rats. Therefore, impairing endogenous VMH CTR-mediated signaling reduced leptin signaling and caused DR rats to become more obese and insulin resistant, both on chow and HFD. These results suggest that endogenous VMH amylin signaling is required for full leptin signaling and protection from HFD-induced obesity.

Role of FAT/CD36 in fatty acid sensing, energy, and glucose homeostasis regulation in DIO and DR rats.

Hypothalamic fatty acid (FA) sensing neurons alter their activity utilizing the FA translocator/receptor, FAT/CD36. Depletion of ventromedial hypothalamus (VMH) CD36 with adeno-associated viral vector expressing CD36 shRNA (AAV CD36 shRNA) leads to redistribution of adipose stores and insulin resistance in outbred rats. This study assessed the requirement of VMH CD36-mediated FA sensing for the regulation of energy and glucose homeostasis in postnatal day 5 (P5) and P21 selectively bred diet-induced obese (DIO) and diet-resistant (DR) rats using VMH AAV CD36 shRNA injections. P5 CD36 depletion altered VMH neuronal FA sensing predominantly in DIO rats. After 10 wk on a 45% fat diet, DIO rats injected with VMH AAV CD36 shRNA at P21 ate more and gained more weight than DIO AAV controls, while DR AAV CD36 shRNA-injected rats gained less weight than DR AAV controls. VMH CD36 depletion increased inguinal fat pad weights and leptin levels in DIO and DR rats. Although DR AAV CD36 shRNA-injected rats became as obese as DIO AAV controls, only DIO control and CD36 depleted rats became insulin-resistant on a 45% fat diet. VMH CD36 depletion stunted linear growth in DIO and DR rats. DIO rats injected with AAV CD36 shRNA at P5 had increased fat mass, mostly due to a 45% increase in subcutaneous fat. They were also insulin-resistant with an associated 71% increase of liver triglycerides. These results demonstrate that VMH CD36-mediated FA sensing is a critical factor in the regulation of energy and glucose homeostasis and fat deposition in DIO and DR rats.

Role of VMH ketone bodies in adjusting caloric intake to increased dietary fat content in DIO and DR rats.

The objective of this study was to determine the potential role of astrocyte-derived ketone bodies in regulating the early changes in caloric intake of diet induced-obese (DIO) versus diet-resistant (DR) rats fed a 31.5% fat high-energy (HE) diet. After 3 days on chow or HE diet, DR and DIO rats were assessed for their ventromedial hypothalamic (VMH) ketone bodies levels and neuronal ventromedial hypothalamic nucleus (VMN) sensing using microdialysis coupled to continuous food intake monitoring and calcium imaging in dissociated neurons, respectively. DIO rats ate more than DR rats over 3 days of HE diet intake. On day 3 of HE diet intake, DR rats reduced their caloric intake while DIO rats remained hyperphagic. Local VMH astrocyte ketone bodies production was similar between DR and DIO rats during the first 6 h after dark onset feeding but inhibiting VMH ketone body production in DR rats on day 3 transiently returned their intake of HE diet to the level of DIO rats consuming HE diet. In addition, dissociated VMN neurons from DIO and DR rats were equally sensitive to the largely excitatory effects of ß-hydroxybutyrate. Thus while DR rats respond to increased VMH ketone levels by decreasing their intake after 3 days of HE diet, this is not the case of DIO rats. These data suggest that DIO inherent leptin resistance prevents ketone bodies inhibitory action on food intake.

Long-term, intermittent, insulin-induced hypoglycemia produces marked obesity without hyperphagia or insulin resistance: a model for weight gain with intensive insulin therapy.

A major side effect of insulin treatment of diabetes is weight gain, which limits patient compliance and may pose additional health risks. Although the mechanisms responsible for this weight gain are poorly understood, it has been suggested that there may be a link to the incidence of recurrent episodes of hypoglycemia. Here we present a rodent model of marked weight gain associated with weekly insulin-induced hypoglycemic episodes in the absence of diabetes. Insulin treatment caused a significant increase in both body weight and fat mass, accompanied by reduced motor activity, lowered thermogenesis in response to a cold challenge, and reduced brown fat uncoupling protein mRNA. However, there was no effect of insulin treatment on total food intake nor on hypothalamic neuropeptide Y or proopiomelanocortin mRNA expression, and insulin-treated animals did not become insulin-resistant. Our results suggest that repeated iatrogenic hypoglycemia leads to weight gain, and that such weight gain is associated with a multifaceted deficit in metabolic regulation rather than to a chronic increase in caloric intake.

Hypothalamic Fkbp51 is induced by fasting, and elevated hypothalamic expression promotes obese phenotypes.

To discover hypothalamic genes that might play a role in regulating energy balance, we carried out a microarray screen for genes induced by a 48-h fast in male C57Bl/6J mouse hypothalamus. One such gene was Fkbp51 (FK506 binding protein 5; Locus NP_034350). The product of this gene is of interest because it blocks glucocorticoid action, suggesting that fasting-induced elevation of this gene in the hypothalamus may reduce glucocorticoid negative feedback, leading to elevated glucocorticoid levels, thus promoting obese phenotypes. Subsequent analysis demonstrated that a 48-h fast induces Fkbp51 in ventromedial, paraventricular, and arcuate hypothalamic nuclei of mice and rats. To assess if hypothalamic Fkbp51 promotes obesity, the gene was transferred to the hypothalamus via an adeno-associated virus vector. Within 2 wk following Fkbp51 overexpression, mice on a high-fat diet exhibited elevated body weight, without hyperphagia, relative to mice receiving the control mCherry vector. Body weight remained elevated for more than 8 wk and was associated with elevated corticosterone and impaired glucose tolerance. These studies suggest that elevated hypothalamic Fkbp51 promotes obese phenotypes.

Erythropoietin-derived peptide treatment reduced neurological deficit and neuropathological changes in a mouse model of tauopathy.

BACKGROUND: Prominent activation of microglial immune/inflammatory processes is a characteristic feature of brains of patients with tauopathies including Alzheimer's disease (AD), suggesting that neuroinflammation may be a critical factor in their pathogenesis. Strategies aimed at developing new therapeutics for tauopathies based on anti-inflammation or immunomodulation are likely to be promising avenues of research. We previously developed JM4-a 19'mer cyclic peptide derived from the first loop of human erythropoietin. This peptide possesses beneficial immune modulatory and tissue protective effects while lacking the undesirable side effects of full-length erythropoietin. In this preclinical study, we investigated the effect of chronic JM4 treatment on the PS19 mouse that carries the P301S mutant human tau gene, linked to a form of frontotemporal dementia. This transgenic mouse has been widely used as a model of tauopathies including AD and related dementias. METHODS: Daily subcutaneous treatment of female PS19 mice with JM4 was initiated before disease onset and continued on for the animals' lifespan. The progression of neurological deficit and the lifespan of these mice were assessed. To evaluate the effect of JM4 treatment on cognition of these animals, the PS19 mice underwent Barnes maze test and elevated plus maze test. In addition, neuronal loss, phosphorylated tau aggregation, and microglial activation were assessed using immunohistochemistry of PS19 mouse brain sections. RESULTS: JM4 treatment of PS19 mice initiated before disease onset reduced neurological deficit, prolonged lifespan, and rescued memory impairment. The beneficial effects of JM4 were accompanied by reductions in neuronal loss, phosphorylated tau aggregation, and microglial activation in the PS19 mouse brain. LIMITATIONS: Use of a single dose of JM4 and female mice only. CONCLUSION: JM4 is a potential novel therapeutic agent for the treatment of tauopathies including AD and related dementias.

Relationship among brain and blood glucose levels and spontaneous and glucoprivic feeding.

Although several studies implicate small declines in blood glucose levels as stimulus for spontaneous meal initiation, no mechanism is known for how these dips might initiate feeding. To assess the role of ventromedial hypothalamus (VMH) (arcuate plus ventromedial nucleus) glucosensing neurons as potential mediators of spontaneous and glucoprivic feeding, meal patterns were observed, and blood and VMH microdialysis fluid were sampled in 15 rats every 10 min for 3.5 h after dark onset and 2 h after insulin (5 U/kg, i.v.) infusion. Blood glucose levels declined by 11% beginning approximately 5 min before 65% of all spontaneous meals, with no fall in VMH levels. After insulin, blood and VMH glucose reached nadirs by 30-40 min, and the same rats ate 60% faster and spent 84% more time eating during the ensuing hypoglycemia. Although 83% of first hypoglycemic meals were preceded by 5 min dips in VMH (but not blood) glucose levels, neither blood nor VMH levels declined before second meals, suggesting that low glucose, rather than changing levels, was the stimulus for glucoprivic meals. Furthermore, altering VMH glucosensing by raising or lowering glucokinase (GK) activity failed to affect spontaneous feeding, body or adipose weights, or glucose tolerance. However, chronic depletion by 26-70% of VMH GK mRNA reduced glucoprivic feeding. Thus, although VMH glucosensing does not appear to be involved in either spontaneous feeding or long-term body-weight regulation, it does participate in glucoprivic feeding, similar to its role in the counter-regulatory neurohumoral responses to glucoprivation.

Effects of maternal genotype and diet on offspring glucose and fatty acid-sensing ventromedial hypothalamic nucleus neurons.

Maternal obesity accentuates offspring obesity in dams bred to develop diet-induced obesity (DIO) on a 31% fat, high-sucrose, high-energy (HE) diet but has no effect on offspring of diet-resistant (DR) dams. Also, only DIO dams become obese when they and DR dams are fed HE diet throughout gestation and lactation. We assessed glucose and oleic acid (OA) sensitivity of dissociated ventromedial hypothalamic nucleus (VMN) neurons from 3- to 4-wk old offspring of DIO and DR dams fed chow or HE diet using fura-2 calcium imaging to monitor intracellular calcium fluctuations as an index of neuronal activity. Offspring of DIO dams fed chow had approximately 2-fold more glucose-inhibited (GI) neurons than did DR offspring. This difference was eliminated in offspring of DIO dams fed HE diet. At 2.5 mM glucose, offspring of chow-fed DIO dams had more GI neurons that were either excited or inhibited by OA than did DR offspring. Maternal HE diet intake generally increased the percentage of neurons that were excited and decreased the percentage that were inhibited by OA in both DIO and DR offspring. However, this effect was more pronounced in DIO offspring. These data, as well as concentration-dependent differences in OA sensitivity, suggest that genotype, maternal obesity, and dietary content can all affect the sensitivity of offspring VMN neurons to glucose and long-chain fatty acids. Such altered sensitivities may underlie the propensity of DIO offspring to become obese when fed high-fat, high-sucrose diets.

Characteristics and mechanisms of hypothalamic neuronal fatty acid sensing.

We assessed the mechanisms by which specialized hypothalamic ventromedial nucleus (VMN) neurons utilize both glucose and long-chain fatty acids as signaling molecules to alter their activity as a potential means of regulating energy homeostasis. Fura-2 calcium (Ca(2+)) and membrane potential dye imaging, together with pharmacological agents, were used to assess the mechanisms by which oleic acid (OA) alters the activity of dissociated VMN neurons from 3- to 4-wk-old rats. OA excited up to 43% and inhibited up to 29% of all VMN neurons independently of glucose concentrations. In those neurons excited by both 2.5 mM glucose and OA, OA had a concentration-dependent effective excitatory concentration (EC(50)) of 13.1 nM. Neurons inhibited by both 2.5 mM glucose and OA had an effective inhibitory concentration (IC(50)) of 93 nM. At 0.5 mM glucose, OA had markedly different effects on these same neurons. Inhibition of carnitine palmitoyltransferase, reactive oxygen species formation, long-chain acetyl-CoA synthetase and ATP-sensitive K(+) channel activity or activation of uncoupling protein 2 (UCP2) accounted for only approximately 20% of OA's excitatory effects and approximately 40% of its inhibitory effects. Inhibition of CD36, a fatty acid transporter that can alter cell function independently of intracellular fatty acid metabolism, reduced the effects of OA by up to 45%. Thus OA affects VMN neuronal activity through multiple pathways. In glucosensing neurons, its effects are glucose dependent. This glucose-OA interaction provides a potential mechanism whereby such "metabolic sensing" neurons can respond to differences in the metabolic states associated with fasting and feeding.

In vivo Bioluminescence Imaging Used to Monitor Disease Activity and Therapeutic Response in a Mouse Model of Tauopathy.

Many studies of tauopathy use transgenic mice that overexpress the P301S mutant form of tau. Neuronal damage in these mice is associated with astrogliosis and induction of glial fibrillary acidic protein (GFAP) expression. GFAP-luc transgenic mice express firefly luciferase under the GFAP promoter, allowing bioluminescence to be measured non-invasively as a surrogate biomarker for astrogliosis. We bred double transgenic mice possessing both P301S and GFAP-luc cassettes and compared them to control mice bearing only the GFAP-luc transgene. We used serial bioluminescent images to define the onset and the time course of astrogliosis in these mice and this was correlated with the development of clinical deficit. Mice containing both GFAP-luc and P301S transgenes showed increased luminescence indicative of astroglial activation in the brain and spinal cord. Starting at 5 months old, the onset of clinical deterioration in these mice corresponded closely to the initial rise in the luminescent signal. Post mortem analysis showed the elevated luminescence was correlated with hyperphosphorylated tau deposition in the hippocampus of double transgenic mice. We used this method to determine the therapeutic effect of JM4 peptide [a small peptide immunomodulatory agent derived from human erythropoietin (EPO)] on double transgenic mice. JM4 treatment significantly decreased the intensity of luminescence, neurological deficit and hyperphosphorylated tau in mice with both the P301S and GFAP-luc transgenes. These findings indicate that bioluminescence imaging (BLI) is a powerful tool for quantifying GFAP expression in living P301S mice and can be used as a noninvasive biomarker of tau-induced neurodegeneration in preclinical therapeutic trials.

Effects of leptin on rat ventromedial hypothalamic neurons.

Neurons in the ventromedial and arcuate hypothalamic nuclei (VMN and ARC, respectively) mediate many of leptin's effects on energy homeostasis. Some are also glucosensing, whereby they use glucose as a signaling molecule to regulate their firing rate. We used fura-2 calcium (Ca2+) imaging to determine the interactions between these two important mediators of peripheral metabolism on individual VMN neurons and the mechanisms by which leptin regulates neuronal activity in vitro. Leptin excited 24%, inhibited 20%, and had a biphasic response in 10% of VMN neurons. Excitation occurred with a EC50 of 5.2 fmol/liter and inhibition with a IC50 of 4.2 fmol/liter. These effects were independent of the ambient glucose levels, and both glucosensing and non-glucosensing neurons were affected by leptin. In contrast, the ARC showed a very different distribution of leptin-responsive neurons, with 40% leptin excited, 10% leptin inhibited, and 2% having a biphasic response (chi2=60.2; P<0.0001). Using pharmacological manipulations we found that leptin inhibits VMN neurons via activation of phosphoinositol-3 kinase and activation of the ATP-sensitive K+ channel. In addition, leptin inhibition was antagonized by 5'-AMP-activated protein kinase activation in 39% of neurons but was unaffected by 5'-AMP-activated protein kinase inhibition. No mechanism was delineated for leptin-induced excitation. Thus, within the physiological range of brain glucose levels, leptin has a differential effect on VMN vs. ARC neurons, and acts on both glucosensing and non-glucosensing VMN neurons in a glucose-independent fashion with inhibition primarily dependent upon activation of the ATP-sensitive K+ channel.

Amylin Selectively Signals Onto POMC Neurons in the Arcuate Nucleus of the Hypothalamus.

Amylin phosphorylates ERK (p-ERK) in the area postrema to reduce eating and synergizes with leptin to phosphorylate STAT3 in the arcuate (ARC) and ventromedial (VMN) hypothalamic nuclei to reduce food intake and body weight. The current studies assessed potential amylin and amylin-leptin ARC/VMN interactions on ERK signaling and their roles in postnatal hypothalamic pathway development. In amylin knockout mice, the density of agouti-related protein (AgRP)-immunoreactive (IR) fibers in the hypothalamic paraventricular nucleus (PVN) was increased, while the density of α-melanocyte-stimulating hormone (αMSH) fibers was decreased. In mice deficient of the amylin receptor components RAMP1/3, both AgRP and αMSH-IR fiber densities were decreased, while only αMSH-IR fiber density was decreased in rats injected neonatally in the ARC/VMN with an adeno-associated virus short hairpin RNA against the amylin core receptor. Amylin induced p-ERK in ARC neurons, 60% of which was present in POMC-expressing neurons, with none in NPY neurons. An amylin-leptin interaction was shown by an additive effect on ARC ERK signaling in neonatal rats and a 44% decrease in amylin-induced p-ERK in the ARC of leptin receptor-deficient and of ob/ob mice. Together, these results suggest that amylin directly acts, through a p-ERK-mediated process, on POMC neurons to enhance ARC-PVN αMSH pathway development.

Altered hypothalamic leptin, insulin, and melanocortin binding associated with moderate-fat diet and predisposition to obesity.

Rats with a genetic predisposition to develop diet-induced obesity (DIO) have a preexisting reduction in central leptin and insulin sensitivity. High-fat diets also reduce sensitivity to leptin, insulin, and melanocortin agonists. We postulated that such reduced sensitivities would be associated with decreased binding to the hypothalamic leptin, insulin, and melanocortin receptors in selectively bred DIO rats and in rats fed a high-energy (HE; 31% fat) diet for 7 wk. On HE diet, DIO rats gained 15% more weight and had 121% heavier fat pads and 70% higher leptin levels than low fat chow-fed DIO rats. Diet-resistant (DR) rats gained no more weight on HE diet but had 48% heavier fat pads and 70% higher leptin levels than chow-fed DR rats. Compared with DR rats, DIO (125)I-leptin binding was 41, 36, and 40% lower in the hypothalamic dorsomedial, arcuate, and dorsomedial portion of the ventromedial nuclei, respectively, and arcuate (125)I-insulin binding was 31% lower independent of diet. In contrast, hypothalamic melanocortin binding did not differ between DIO and DR rats. However, HE diet intake lowered lateral hypothalamic melanocortin-3 and melanocortin-4 receptor and hippocampal insulin binding of both DIO and DR rats and hypothalamic paraventricular nucleus melanocortin-4 receptor binding only in DR rats. Neither genotype nor diet affected substantia nigra or ventral tegmental area binding. These results corroborate our previous findings demonstrating a preexisting decrease in DIO hypothalamic leptin and insulin signaling and demonstrate that HE diet intake reduces hypothalamic melanocortin and hippocampal insulin binding.

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.

Cortical Fluoro-Jade staining and blunted adrenomedullary response to hypoglycemia after noncoma hypoglycemia in rats.

Intensive insulin therapy in patients with type 1 diabetes mellitus reduces long-term complications; however, intensive therapy is also associated with a three-fold increase in hypoglycemic episodes. The present study in conscious rats characterizes the physiologic and neuropathologic consequences of a single episode of moderate hypoglycemia. In this model, intravenous insulin is used to reduce plasma glucose to 30 to 35 mg/dL for 75 mins. This single hypoglycemic insult acutely induces hypoglycemia-associated autonomic failure (HAAF), with epinephrine responses to hypoglycemia reduced more than 36% from control. Neuropathology after this insult includes the appearance of dying cells, assessed with the marker Fluoro-jade B (FJ). After hypoglycemic insult, FJ+ cells were consistently seen in subdivisions of the medial prefrontal cortex, the orbital cortex, and the piriform cortex. There was a significant correlation between depth of hypoglycemia and number of FJ+ cells, suggesting that there is a critical threshold below which vulnerable cells begin to die. These data suggest that there is a population of cells that are vulnerable to moderate levels of hypoglycemia commonly experienced by patients with insulin-treated diabetes. These cells, which may be neurons, are primarily found in cortical regions implicated in visceral perception and autonomic control, raising the possibility that their loss contributes to clinically reported deficits in autonomic and perceptual responses to hypoglycemia.

Third ventricular alloxan reversibly impairs glucose counterregulatory responses.

Glucokinase (GK) is hypothesized to be the critical glucosensor of pancreatic beta-cells and hypothalamic glucosensing neurons. To understand the role of GK in glucoprivic counterregulatory responses, we injected alloxan, a GK inhibitor and toxin, into the third ventricle (3v) to target nearby GK-expressing neurons. Four and 6 days after 3v, but not 4v, alloxan injection, alloxan-treated rats ate only 30% and their blood glucose area under the curve was only 28% of saline controls' after systemic 2-deoxy-D-glucose. In addition, their hyperglycemic response to hindbrain glucoprivation induced with 5-thio-glucose was impaired, whereas fasting blood glucose levels and food intake after an overnight fast were elevated. These impaired responses were associated with the destruction of 3v tanycytes, reduced glial fibrillary acidic protein-immunoreactivity surrounding the 3v, neuronal swelling, and decreased arcuate nucleus neuropeptide Y (NPY) mRNA. Nevertheless, hypothalamic GK mRNA was significantly elevated. Two weeks after alloxan injection, 3v tanycyte destruction was reversed along with restoration of feeding and hyperglycemic responses to both systemic and hindbrain glucoprivation. At this time there were significant decreases in GK, NPY, and proopiomelanocortin mRNA. Thus, neural substrates near and around the 3v affected by alloxan may be critically involved in the expression of these glucoprivic responses.

Glucokinase is the likely mediator of glucosensing in both glucose-excited and glucose-inhibited central neurons.

Specialized neurons utilize glucose as a signaling molecule to alter their firing rate. Glucose-excited (GE) neurons increase and glucose-inhibited (GI) neurons reduce activity as ambient glucose levels rise. Glucose-induced changes in the ATP-to-ADP ratio in GE neurons modulate the activity of the ATP-sensitive K(+) channel, which determines the rate of cell firing. The GI glucosensing mechanism is unknown. We postulated that glucokinase (GK), a high-Michaelis constant (K(m)) hexokinase expressed in brain areas containing populations of GE and GI neurons, is the controlling step in glucosensing. Double-label in situ hybridization demonstrated neuron-specific GK mRNA expression in locus ceruleus norepinephrine and in hypothalamic neuropeptide Y, pro-opiomelanocortin, and gamma-aminobutyric acid neurons, but it did not demonstrate this expression in orexin neurons. GK mRNA was also found in the area postrema/nucleus tractus solitarius region by RT-PCR. Intracarotid glucose infusions stimulated c-fos expression in the same areas that expressed GK. At 2.5 mmol/l glucose, fura-2 Ca(2+) imaging of dissociated ventromedial hypothalamic nucleus neurons demonstrated GE neurons whose intracellular Ca(2+) oscillations were inhibited and GI neurons whose Ca(2+) oscillations were stimulated by four selective GK inhibitors. Finally, GK expression was increased in rats with impaired central glucosensing (posthypoglycemia and diet-induced obesity) but was unaffected by a 48-h fast. These data suggest a critical role for GK as a regulator of glucosensing in both GE and GI neurons in the brain.