2DG Administration Research Articles

Raphe glucose-sensing serotonergic neurons stimulate KNDy neurons to enhance LH pulses via 5HT2CR: rat and goat studies

Dysfunction of central serotonergic neurons is known to cause depressive disorders in humans, who often show reproductive and/or glucose metabolism disorders. This study examined whether dorsal raphe (DR) serotonergic neurons sense high glucose availability to upregulate reproductive function via activating hypothalamic arcuate (ARC) kisspeptin neurons (= KNDy neurons), a dominant stimulator of gonadotropin-releasing hormone (GnRH)/gonadotropin pulses, using female rats and goats. RNA-seq and histological analysis revealed that stimulatory serotonin-2C receptor (5HT2CR) was mainly expressed in the KNDy neurons in female rats. The serotonergic reuptake inhibitor administration into the mediobasal hypothalamus (MBH), including the ARC, significantly blocked glucoprivic suppression of luteinizing hormone (LH) pulses and hyperglycemia induced by intravenous 2-deoxy-D-glucose (2DG) administration in female rats. A local infusion of glucose into the DR significantly increased in vivo serotonin release in the MBH and partly restored LH pulses and hyperglycemia in the 2DG-treated female rats. Furthermore, central administration of serotonin or a 5HT2CR agonist immediately evoked GnRH pulse generator activity, and central 5HT2CR antagonism blocked the serotonin-induced facilitation of GnRH pulse generator activity in ovariectomized goats. These results suggest that DR serotonergic neurons sense high glucose availability to reduce gluconeogenesis and upregulate reproductive function by activating GnRH/LH pulse generator activity in mammals.

Enkephalin-δ Opioid Receptor Signaling Mediates Glucoprivic Suppression of LH Pulse and Gluconeogenesis in Female Rats.

Energy availability is an important regulator of reproductive function at various reproductive phases in mammals. Glucoprivation induced by 2-deoxy-D-glucose (2DG), an inhibitor of glucose utilization, as an experimental model of malnutrition suppresses the pulsatile release of GnRH/LH and induces gluconeogenesis. The present study was performed with the aim of examining whether enkephalin-δ-opioid receptor (DOR) signaling mediates the suppression of pulsatile GnRH/LH release and gluconeogenesis during malnutrition. The administration of naltrindole hydrochloride (NTI), a selective DOR antagonist, into the third ventricle blocked the suppression of LH pulses and part of gluconeogenesis induced by IV 2DG administration in ovariectomized rats treated with a negative feedback level of estradiol-17 β (OVX + low E2). The IV 2DG administration significantly increased the number of Penk (enkephalin gene)-positive cells coexpressing fos (neuronal activation marker gene) in the paraventricular nucleus (PVN), but not in the arcuate nucleus (ARC) in OVX + low E2 rats. Furthermore, double in situ hybridization for Penk/Pdyn (dynorphin gene) in the PVN revealed that approximately 35% of the PVN Penk-expressing cells coexpressed Pdyn. Double in situ hybridization for Penk/Crh (corticotropin-releasing hormone gene) in the PVN and Penk/Kiss1 (kisspeptin gene) in the ARC revealed that few Penk-expressing cells coexpressed Crh and Kiss1. Taken together, these results suggest that central enkephalin-DOR signaling mediates the suppression of pulsatile LH release during malnutrition. Moreover, the current study suggests that central enkephalin-DOR signaling is also involved in gluconeogenesis during malnutrition in female rats.

Endogenous Androgens Diminish Food Intake and Activation of Orexin A Neurons in Response to Reduced Glucose Availability in Male Rats.

Sex steroids modify feeding behavior and body weight regulation, and androgen reportedly augments food intake and body weight gain. To elucidate the role of endogenous androgens in the feeding regulation induced by reduced glucose availability, we examined the effect of gonadectomy (orchiectomy) on food intake and orexin A neuron’s activity in the lateral hypothalamic/perifornical area (LH/PFA) in response to reduced glucose availability (glucoprivation) induced by 2-deoxy-d-glucose (2DG) administration in male rats. Rats (7W) were bilaterally orchiectomized (ORX group) or sham operated (Sham group). Seventeen days after the surgery, food intake response to 2DG (400 mg/kg, i.v.) was measured for 4 h after the infusion. The same experiment was performed for the immunohistochemical examination of c-Fos-expressing orexin A neurons in the LH/PFA and c-Fos expression in the arcuate nucleus (Arc). Food intake induced by glucoprivation was greater in the ORX group than the Sham group, and the glucoprivation-induced food intake was inversely correlated with plasma testosterone concentration. Glucoprivation stimulated c-Fos expression of the orexin A neurons at the LH/PFA and c-Fos expression in the dorsomedial Arc. The number and percentage of c-Fos-expressing orexin A neurons in the LH/PFA and c-Fos expression in the dorsomedial Arc were significantly higher in the ORX group than the Sham group. This indicates that endogenous androgen, possibly testosterone, diminishes the food intake induced by reduced glucose availability, possibly via the attenuated activity of orexin A neuron in the LH/PFA and neurons in the dorsomedial Arc.

Attenuation of the Counter-Regulatory Glucose Response in CVLM C1 Neurons: A Possible Explanation for Anorexia of Aging

This study aimed to determine the effect of age on CVLM C1 neuron glucoregulatory proteins in the feeding pathway. Male Sprague Dawley rats aged 3 months and 24 months old were divided into two subgroups: the treatment group with 2-deoxy-d-glucose (2DG) and the control group. Rat brains were dissected to obtain the CVLM region of the brainstem. Western blot was used to determine protein expression of tyrosine hydroxylase (TH), phosphorylated TH at Serine40 (pSer40TH), AMP-activated protein kinase (AMPK), phosphorylated AMPK (phospho AMPK), and neuropeptide Y Y5 receptors (NPY5R) in CVLM samples. Immunofluorescence was used to determine TH-, AMPK-, and NPY5R-like immunoreactivities among other brain coronal sections. Results obtained denote a decrease in basal TH phosphorylation levels and AMPK proteins and an increase in TH proteins among aged CVLM neurons. Increases in the basal immunoreactivity of TH+, AMPK+, NPY5R+, TH+/AMPK+, and TH+/NPY5R+ were also observed among old rats. Young treatment-group rats saw a decrease in TH phosphorylation and AMPK proteins following 2DG administration, while an increase in AMPK phosphorylation and a decrease in TH proteins were found among the old-treatment-group rats. These findings suggest the participation of CVLM C1 neurons in counter-regulatory responses among young and old rats. Altering protein changes in aged CVLM C1 neurons may attenuate responses to glucoprivation, thus explaining the decline in food intake among the elderly.

Central µ-Opioid Receptor Antagonism Blocks Glucoprivic LH Pulse Suppression and Gluconeogenesis/Feeding in Female Rats.

Energetic status often affects reproductive function, glucose homeostasis, and feeding in mammals. Malnutrition suppresses pulsatile release of the gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) and increases gluconeogenesis and feeding. The present study aims to examine whether β-endorphin-μ-opioid receptor (MOR) signaling mediates the suppression of pulsatile GnRH/LH release and an increase in gluconeogenesis/feeding induced by malnutrition. Ovariectomized female rats treated with a negative feedback level of estradiol-17β (OVX + low E2) receiving 2-deoxy-D-glucose (2DG), an inhibitor of glucose utilization, intravenously (iv) were used as a malnutrition model. An administration of D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), a selective MOR antagonist, into the third ventricle blocked the suppression of the LH pulse and increase in gluconeogenesis/feeding induced by iv 2DG administration. Histological analysis revealed that arcuate Kiss1 (kisspeptin gene)-expressing cells and preoptic Gnrh1 (GnRH gene)-expressing cells co-expressed little Oprm1 (MOR gene), while around 10% of arcuate Slc17a6 (glutamatergic marker gene)-expressing cells co-expressed Oprm1. Further, the CTOP treatment decreased the number of fos-positive cells in the paraventricular nucleus (PVN) in OVX + low E2 rats treated with iv 2DG but failed to affect the number of arcuate fos-expressing Slc17a6-positive cells. Taken together, these results suggest that the central β-endorphin-MOR signaling mediates the suppression of pulsatile LH release and that the β-endorphin may indirectly suppress the arcuate kisspeptin neurons, a master regulator for GnRH/LH pulses during malnutrition. Furthermore, the current study suggests that central β-endorphin-MOR signaling is also involved in gluconeogenesis and an increase in food intake by directly or indirectly acting on the PVN neurons during malnutrition in female rats.

Paraventricular Dynorphin A Neurons Mediate LH Pulse Suppression Induced by Hindbrain Glucoprivation in Female Rats.

Malnutrition suppresses reproductive functions in mammals, which is considered to be mostly due to the inhibition of pulsatile gonadotropin-releasing hormone (GnRH)/gonadotropin secretion. Accumulating evidence suggests that kisspeptin neurons in the arcuate nucleus (ARC) play a critical role in the regulation of pulsatile GnRH/gonadotropin release. The present study aimed to examine if the hypothalamic dynorphin A (Dyn) neurons mediate the suppression of GnRH/luteinizing hormone (LH) pulses during malnutrition. Ovariectomized rats treated with a negative feedback level of estradiol-17β-treated (OVX+E2) were administered with intravenous (iv) or fourth cerebroventricle (4V) 2-deoxy-D-glucose (2DG), an inhibitor of glucose utilization, to serve as a malnutrition model. Central administration of a Dyn receptor antagonist blocked the iv- or 4V-2DG-induced suppression of LH pulses in OVX+E2 rats. The 4V 2DG administration significantly increased the number of Pdyn (Dyn gene)-positive cells co-expressing fos in the paraventricular nucleus (PVN), but not in the ARC and supraoptic nucleus (SON), and the iv 2DG treatment significantly increased the number of fos and Pdyn-co-expressing cells in the PVN and SON, but decreased it in the ARC. The E2 treatment significantly increased Pdyn expression in the PVN, but not in the ARC and SON. Double in situ hybridization for Kiss1 (kisspeptin gene) and Oprk1 (Dyn receptor gene) revealed that around 60% of ARC Kiss1-expressing cells co-expressed Oprk1. These results suggest that the PVN Dyn neurons, at least in part, mediate LH pulse suppression induced by the hindbrain or peripheral glucoprivation, and Dyn neurons may directly suppress the ARC kisspeptin neurons in female rats.

Effects of age on feeding response: Focus on the rostral C1 neuron and its glucoregulatory proteins.

Older people are likely to develop anorexia of aging. Rostral C1 (rC1) catecholaminergic neurons in rostral ventrolateral medulla (RVLM) are recently discovered its role in food intake control. It is well established that these neurons regulate cardiovascular function. This study aims to determine the effect of age on the function of rostral C1 (rC1) neurons in mediating feeding response. Male Sprague Dawley rats at 3-months (n = 22) and 24-months (n = 22) old were used and further divided into two subgroups; 1) treatment group with 2-deoxy-d-glucose (2DG) and 2) vehicle group. Feeding hormones such as cholecystokinin (CCK), ghrelin and leptin were analysed using enzyme-linked immunosorbent assay (ELISA). Rat brain was carefully dissected to obtain the brainstem RVLM region. Further analysis was carried out to determine the level of proteins and genes in RVLM that were associated with feeding pathway. Protein expression of tyrosine hydroxylase (TH), phosphorylated TH at Serine40 (pSer40TH), AMP-activated protein kinase (AMPK), phosphorylated AMPK (phospho AMPK) and neuropeptide Y Y5 receptor (NPY5R) were determined by western blot. Expression of TH, AMPK and NPY genes were determined by real-time PCR. This study showed that blood glucose level was elevated in young and old rats following 2DG administration. Plasma CCK-8 concentration was higher in the aged rats at basal and increased with 2DG administration in young rats, but the leptin and ghrelin showed no changes. Old rats showed higher TH and lower AMPK mRNA levels. Glucoprivation decreased AMPK mRNA level in young rats and decreased TH mRNA in old rats. Aged rC1 neurons showed higher NPY5R protein level. Following glucoprivation, rC1 neurons produced distinct molecular changes across age in which, in young rats, AMPK phosphorylation level was increased and in old rats, TH phosphorylation level was increased. These findings suggest that glucose-counterregulatory responses by rC1 neurons at least, contribute to the ability of young and old rats in coping glucoprivation. Age-induced molecular changes within rC1 neurons may attenuate the glucoprivic responses. This situation may explain the impairment of feeding response in the elderly.

Growth hormone enhances the recovery of hypoglycemia via ventromedial hypothalamic neurons.

Growth hormone (GH) is secreted during hypoglycemia, and GH-responsive neurons are found in brain areas containing glucose-sensing neurons that regulate the counter-regulatory response (CRR). However, whether GH modulates the CRR to hypoglycemia via specific neuronal populations is currently unknown. Mice carrying ablation of GH receptor (GHR) either in leptin receptor (LepR)- or steroidogenic factor-1 (SF1)-expressing cells were studied. We also investigated the importance of signal transducer and activator of transcription 5 (STAT5) signaling in SF1 cells for the CRR. GHR ablation in LepR cells led to impaired capacity to recover from insulin-induced hypoglycemia and to a blunted CRR caused by 2-deoxy-d-glucose (2DG) administration. GHR inactivation in SF1 cells, which include ventromedial hypothalamic neurons, also attenuated the CRR. The reduced CRR was prevented by parasympathetic blockers. Additionally, infusion of 2DG produced an abnormal hyperactivity of parasympathetic preganglionic neurons, whereas the 2DG-induced activation of anterior bed nucleus of the stria terminalis neurons was reduced in mice without GHR in SF1 cells. Mice carrying ablation of Stat5a/b genes in SF1 cells showed no defects in the CRR. In summary, GHR expression in SF1 cells is required for a normal CRR, and these effects are largely independent of STAT5 pathway.-Furigo, I. C., de Souza, G. O., Teixeira, P. D. S., Guadagnini, D., Frazão, R., List, E. O., Kopchick, J. J., Prada, P. O., Donato, J., Jr. Growth hormone enhances the recovery of hypoglycemia via ventromedial hypothalamic neurons.

Combining De-Glycosylating Agents with CAR-T Cells for Targeting Solid Tumors and Reducing Toxicity

▪Background: The adoptive transfer of CAR-T cells have shown impressive results against B-cell malignancies, but still limited efficacy against solid tumors. The discovery of the key factors regulating the activity of CAR-T cells is required to improve their antitumor potency and modulate toxicities. Since solid tumors display a wide range of glycosylation alterations, including increased N-glycan branching, we hypothesized that peptidic epitopes may be masked by glycans from CAR-T cell targeting, especially in richly glycosylated proteins.Results: To investigate if sugar chains may be sterically hulking for CAR-T cell targeting, we generated N-glycosylation-defective pancreatic tumor cell lines. This aim has been achieved by knocking-out the expression of the glycosyltransferase Mgat5, a key enzyme involved in the process of N-glycan branching, using the CRISPR-Cas9 technology. As model antigens for CAR targeting, we focused on CD44v6 and CEACAM-5 (CEA) since they are both heavily glycosylated proteins over-expressed on a wide variety of solid tumors, including pancreatic adenocarcinoma. Strikingly, the impairment of N-glycosylation resulted in a dramatic increase of tumor targeting by both CD44v6 (4-fold, p<0,001) and CEA CAR-T cells (10-fold, p<0,001). This effect associated with improved CAR-T cell activation, suggesting more proficient antigen engagement. To exploit this mechanism in order to increase the efficacy of CAR-T cells against solid tumors, we sought to block tumor N-glycosylation with the clinical-grade glucose/mannose analogue 2-Deoxy-D-glucose (2DG). Similarly to genetically induced glycosylation blockade, treatment with 2DG also sensitized tumor cells to recognition by CAR-T cells, significantly increasing their elimination (CD44v6: 3-fold, p<0,01; CEA: 13-fold, p<0,001). Notably, 2DG alone proved to be ineffective as mono-therapy, suggesting a synergistic effect with CAR-T cells. To get more insights on this mechanism, we took advantage of previous studies reporting that 2DG interference with N-glycosylation can be reverted by the addition of exogenous mannose. Of notice, mannose did revert the synergy between 2DG and CAR-T cells (p<0,05), implying that blockade of N-glycosylation rather than glycolysis is the crucial mechanism involved. These findings were further confirmed by using the N-glycosylation inhibitor tunicamycin (CD44v6: 2,5-fold; CEA: 5-fold, p<0,01) and by Western blot, looking at the presence of de-glycosylated proteins on tumor cell surface after 2DG treatment. Next, we challenged the combined approach in a pancreatic adenocarcinoma xenograft mouse model. Accordingly with in vitro data, mice receiving CAR-T cells highly benefited from 2DG administration (5-fold less tumor at 7d, p<0,05), which conversely was unable to mediate any antitumor effect alone. Interestingly, improved antitumor activity was accompanied by a decrease in the frequency of CAR-T cells expressing one or more exhaustion and senescence markers, such as TIM-3, LAG-3, PD-1 and CD57 (SPICE software analysis, p=0,0105). Finally, thanks to metabolic deregulation (Warburg effect), 2DG is expected to selectively accumulate in cancer cells compared to healthy tissues, supporting the safety of the combined approach. Accordingly, we observed that the same doses of 2DG able to enhance tumor cell recognition by CAR-T cells failed to increase the elimination of healthy cells, such as keratinocytes.Conclusions: Our results indicate that i) the glycosylation status of tumor cells regulates the efficacy of CAR-T cells, especially when targeting highly glycosylated antigens, and ii) combining CAR-T cells with the de-glycosylation agent 2DG, which preferentially accumulates in tumor masses, may pave the way for a successful immunotherapy against solid tumors. DisclosuresBonini:Intellia Therapeutics: Research Funding. Bondanza:Novartis: Employment.

The Suprachiasmatic Nucleus Modulates the Sensitivity of Arcuate Nucleus to Hypoglycemia in the Male Rat.

The suprachiasmatic nucleus (SCN) and arcuate nucleus (ARC) have reciprocal connections; catabolic metabolic information activates the ARC and inhibits SCN neuronal activity. Little is known about the influence of the SCN on the ARC. Here, we investigated whether the SCN modulated the sensitivity of the ARC to catabolic metabolic conditions. ARC neuronal activity, as determined by c-Fos immunoreactivity, was increased after a hypoglycemic stimulus by 2-deoxyglucose (2DG). The highest ARC neuronal activity after 2DG was found at the end of the light period (zeitgeber 11, ZT11) with a lower activity in the beginning of the light period (zeitgeber 2, ZT2), suggesting the involvement of the SCN. The higher activation of ARC neurons after 2DG at ZT11 was associated with higher 2DG induced blood glucose levels as compared with ZT2. Unilateral SCN-lesioned animals, gave a mainly ipsilateral activation of ARC neurons at the lesioned side, suggesting an inhibitory role of the SCN on ARC neurons. The 2DG-induced counterregulatory glucose response correlated with increased ARC neuronal activity and was significantly higher in unilateral SCN-lesioned animals. Finally, the ARC as site where 2DG may, at least partly, induce a counterregulatory response was confirmed by local microdialysis of 2DG. 2DG administration in the ARC produced a higher increase in circulating glucose compared with 2DG administration in surrounding areas such as the ventromedial nucleus of the hypothalamus (VMH). We conclude that the SCN uses neuronal pathways to the ARC to gate sensory metabolic information to the brain, regulating ARC glucose sensitivity and counterregulatory responses to hypoglycemic conditions.

Mapping brain glucose uptake with chemical exchange-sensitive spin-lock magnetic resonance imaging.

Uptake of administered D-glucose (Glc) or 2-deoxy-D-glucose (2DG) has been indirectly mapped through the chemical exchange (CE) between glucose hydroxyl and water protons using CE-dependent saturation transfer (glucoCEST) magnetic resonance imaging (MRI). We propose an alternative technique—on-resonance CE-sensitive spin-lock (CESL) MRI—to enhance responses to glucose changes. Phantom data and simulations suggest higher sensitivity for this ‘glucoCESL' technique (versus glucoCEST) in the intermediate CE regime relevant to glucose. Simulations of CESL signals also show insensitivity to B0-fluctuations. Several findings are apparent from in vivo glucoCESL studies of rat brain at 9.4 Tesla with intravenous injections. First, dose-dependent responses are nearly linearly for 0.25-, 0.5-, and 1-g/kg Glc administration (obtained with 12-second temporal resolution), with changes robustly detected for all doses. Second, responses at a matched dose of 1 g/kg are much larger and persist for a longer duration for 2DG versus Glc administration, and are minimal for mannitol as an osmolality control. And third, with similar increases in steady-state blood glucose levels, glucoCESL responses are ∼2.2 times higher for 2DG versus Glc, consistent with their different metabolic properties. Overall, we show that glucoCESL MRI could be a highly sensitive and quantifiable tool for glucose transport and metabolism studies.

Orchiectomy increases food intake and perifornical orexin A neuron's activity during glucoprivation

We examined the effect of orchiectomy on food intake and lateral hypothalamic neuron fs activity during glucoprivation induced by i.v. 2‐deoxy‐D‐glucose (2DG) administration in male rats. Rats (7W) were bilaterally orchiectomized (OrX) or sham‐operated (Sham). 2DG (400 mg/kg) was infused two weeks after the operation and food intake was measured for 3 hours after the injection. The same experiment was performed for immunohistochemical examination of c‐Fos and orexin A expression in the perifornical area (PFA) and arcuate nucleus (Arc). Glucoprivation induced food intake, and food intake in OrX rats was larger than Sham rats, and food intake was inversely correlated with plasma testosterone concentration. Glucoprivation stimulated c‐Fos expression in the orexin A neurons at the PFA, and c‐Fos expression in the Arc. The number and fraction of c‐Fos expressed orexin A neurons was significantly higher in OrX rats than Sham rats. The number of c‐Fos ir cells in the Arc was also higher in OrX rats than Sham rats. These data indicate that testosterone attenuates food intake induced by reduced glucose availability via attenuated perifornical orexin A neuron's activity and Arc neuron's activity.

A phase I dose-escalation trial of 2-deoxy-d-glucose alone or combined with docetaxel in patients with advanced solid tumors

This phase I trial was initiated to evaluate the safety, pharmacokinetics (PK) and maximum tolerated dose (MTD) of the glycolytic inhibitor, 2-deoxy-D-glucose (2DG) in combination with docetaxel, in patients with advanced solid tumors. A modified accelerated titration design was used. 2DG was administered orally once daily for 7 days every other week starting at a dose of 2 mg/kg and docetaxel was administered intravenously at 30 mg/m(2) for 3 of every 4 weeks beginning on day 1 of week 2. Following the completion of dose escalation, cohorts of patients were then treated with 2DG for 21 days or every day of each 4-week cycle for up to 12 cycles. Thirty-four patients were enrolled: 21 on every other week, 6 on a 21 of 28-day cycle and 7 on the continuous 2DG dosing schedule. There were no dose-limiting toxicities which met the MTD criteria. The most common adverse events were fatigue, sweating, dizziness and nausea mimicking the hypoglycemic symptoms expected from 2DG administration. Therefore, 63 mg/kg was selected as the clinically tolerable dose. The most significant adverse effects noted at 63-88 mg/kg doses were reversible hyperglycemia (100 %), gastrointestinal bleeding (6 %) and reversible grade 3 QTc prolongation (22 %). Eleven patients (32 %) had stable disease, 1 patient (3 %) partial response and 22 patients (66 %) progressive disease as their best response. There was no PK interaction between 2DG and docetaxel. The recommended dose of 2DG in combination with weekly docetaxel is 63 mg/kg/day with tolerable adverse effects.

Involvement of orexin A and melanin concentrating hormone neurons of the lateral hypothalamic area in the food intake response to reduced glucose availability

The purpose of the present study was to elucidate the involvement of melanin concentrating hormone (MCH) and orexin A neurons at the lateral hypothalamic area (LHA) in the regulation of food intake stimulated by reduced glucose availability. In the present study, we examined the food intake response and c‐Fos expression in the MCH and orexin A neurons after 2‐deoxy‐D‐glucose (2DG)‐induced glucoprivation or systemic insulin‐induced hypoglycemia. Nine‐week‐old male rats were injected with either 2‐deoxy‐D‐glucose (2DG; 400 mg/kg; i.v.) or insulin (5 units/kg; s.c.), and food and water intakes were measured. Two days after the behavioral test, we examined c‐Fos expression in the MCH and orexin A neurons in the LHA immunohistochemically after 2DG or insulin administration. Both MCH and orexin A neurons were located in the LHA, but these peptides were distinctly localized in the separate neurons. Both 2DG and insulin administrations increased food intake compared with saline administration, and induced c‐Fos expression in the orexin A neurons, but did not induce c‐Fos expression in the MCH neurons of the LHA. These data indicate that orexin A neurons play a role but MCH neurons are not involved in the food intake response to reduced glucose availability.

Estrogen attenuates food intake and perifornical orexinergic neuron′s activity during glucoprivation in ovariectomized rats

We examined the effect of estrogen (E2) replacement in ovariectomized rats on food intake and lateral hypothalamic neuron′s activity during glucoprivation induced by i.v. 2‐deoxy‐D‐glucose (2DG) administration. Rats were ovariectomized and implanted E2 or vehicle (cholesterol) subcutaneously. Two weeks after the replacement, rats were infused with 2DG (400 mg/kg) and food intake was measured for 3 hours. The same experiment was performed for immunohistochemical examination of c‐Fos, melanin concentrating hormone (MCH), and orexin A expression in the lateral hypothalamic area (LH) and arcuate nucleus (Arc). Glucoprivation induced c‐Fos expression in the orexin A neurons locating at the perifornical region of LH, but did not activate MCH neurons. Glucoprivation significantly induced food intake, and food intake in E2 replaced group was significantly lower than E2 deficit group. The number and fraction of c‐Fos expressed orexinergic neurons was significantly lower in E2 replaced group than E2 deficit group. The number of c‐Fos ir cells was less in E2 replaced group than E2 deficit rats. These data indicate that E2‐induced reduction of food intake induced by 2DG is possibly due to the reduced perifornical orexinergic neuron′s activity and to the reduced Arc neuron′s activity.

Anterograde transneuronal viral tract tracing reveals central sensory circuits from white adipose tissue

The origins of the sympathetic nervous system (SNS) innervation of white adipose tissue (WAT) have been defined using the transneuronal viral retrograde tract tracer, pseudorabies virus. Activation of this SNS innervation is acknowledged as the principal initiator of WAT lipolysis. The central control of WAT lipolysis may require neural feedback to a brain-SNS-WAT circuit via WAT afferents. Indeed, conventional tract tracing studies have demonstrated that peripheral pseudounipolar dorsal root ganglion (DRG) sensory cells innervate WAT. The central nervous system projections of WAT afferents remain uncharted, however, and form the focus of the present study. We used the H129 strain of the herpes simplex virus-1 (HSV-1), an anterograde transneuronal viral tract tracer, to define the afferent circuits projecting from WAT to the central nervous system. Siberian hamster inguinal (IWAT) or epididymal WAT was injected with H129 and the neuraxis processed for HSV-1 immunoreactivity. We found substantial overlap in the pattern of WAT sensory afferent projections with multiple SNS outflow sites along the neuraxis, suggesting the possibility of WAT sensory-SNS circuits that could regulate WAT SNS drive and thereby lipolysis. Previously, we demonstrated that systemic 2-deoxy-d-glucose (2DG) elicited increases in the SNS drive to IWAT. Here, we show that systemic 2DG administration also significantly increases multiunit spike activity arising from decentralized IWAT afferents. Collectively, these data provide structural and functional support for the existence of a sensory WAT pathway to the brain, important in the negative feedback control of lipid mobilization.

Central but not peripheral glucoprivation is impaired in monosodium glutamate-treated rats

In the present study, newborn male Wistar rats were injected, subcutaneously, five times, every other day, with monosodium glutamate (MSG, 4 g/kg bw) or saline (as control, C), during the neonatal period. MSG animals developed destruction of the arcuate nuclei (ARC) with absence of NPY-immunoreactive cell bodies, which impaired both the food intake (baseline) and the 2-deoxy- d-glucose (2DG) glucoprivic feeding response. Increases in the immunoreactivity of corticotropin-releasing hormone-cell bodies in the paraventricular nuclei might have developed to compensate for the atrophy of the pituitary in MSG-treated rats. After systemic 2DG injection, neither the C nor the MSG rats increased their food intake, but they showed similar hyperglycemic responses, whereas plasma free fatty acids (FFA) increased only in the C group. In other groups, 2DG, norepinephrine (NE), neostigmine (NEO) and saline were intracerebroventricularly (i.c.v.) administered. In this condition, impairment of the hyperglycemic and food intake responses, associated to a lower increase in plasma FFA levels, were observed. As opposed to this, the MSG treatment gives support to NE effects, enhancing food intake, as well as plasma glucose and FFA levels. After NEO, plasma glucose increased only in the MSG group, while plasma FFA levels were elevated in the C rats. Taken together, the results obtained after MSG treatment point to a separate neural control of the hyperglycemic response and of the lipid mobilization when stimulated by central 2DG, NE or NEO administration. It seems likely that the excitatory neural pathway that controls lipid metabolism and is present in C rats was destroyed by the MSG treatment.

Orexin A-induced feeding is augmented by caloric challenge

Orexin neurons are stimulated by conditions that are glucoprivic, suggesting that orexin signaling may be increased during nutritional duress. We have previously shown that injection of orexin A (OxA) into the rostral lateral hypothalamic area (rLHa) robustly and dose-dependently increases feeding behavior. Thus we hypothesized that exogenous administration of orexin A would induce a greater feeding response after acute food deprivation or perceived caloric duress achieved through 2-deoxyglucose (2DG) administration. To test our hypothesis, male Sprague-Dawley rats implanted with internal guide cannulas directed to the rLHa were exposed to varying degrees of food deprivation (0, 3, 12, 24 h) and 2DG (200 mg/kg) before intra-rLHa OxA (500 pmol) infusion. We also performed a dose-response study using graded doses of OxA (0, 31.25, 125, and 500 pmol) in fed and 24-h fasted rats. OxA administration in conjunction with the highest level of prior food deprivation (24 h) resulted in the greatest feeding response (above baseline means; 0 h deprivation: 1.9 +/- 0.6; 24 h deprivation: 4.4 +/- 0.8; P = 0.0034) and showed a dose-dependent enhancement of feeding. Additionally, 2DG administration before OxA administration resulted in a significantly higher feeding response (above baseline means: 2DG = 1.8 +/- 0.5; OxA = 1.8 +/- 0.4; 2DG + OxA = 5.1 +/- 0.6; P < 0.0001). These data support the hypothesis that orexin signaling may be important in modulating the feeding network under times of nutritional duress.

Glucose uptake in vivo in skeletal muscles of insulin-injected chicks

Glucose uptake across the plasma membrane in animal cells plays a crucial role in whole-body glucose homeostasis. Insulin-stimulated glucose transport activity in vivo in several tissues was estimated using the 2-deoxy- d-[1- 3H]glucose ([ 3H]2DG) uptake determination method. A tracer dose of [ 3H]2DG was injected intravenously into 8-day-old chicks ( Gallus gallus) administered simultaneously or previously with porcine insulin (40 μg/kg BW). After 10 or 20 min, several major tissues, including skeletal and cardiac muscle, were sampled and their 2-deoxy- d-[1- 3H]glucose 6-phosphate content analyzed. Plasma glucose concentration and [ 3H]2DG radioactivity were lowered by insulin within 20 min of [ 3H]2DG administration, while the plasma [ 3H]2DG/glucose ratio was not significantly different between chicks injected with insulin and their control counterparts. A marked uptake of 2DG was observed in cardiac tissue and brain, followed by kidney and skeletal muscles. In skeletal muscles, insulin increased the 2DG uptake in soleus, extensor digitorum longus and pectoralis superficialis muscles. On the other hand, no significant increases in insulin-induced 2DG uptake were detected in cardiac muscle or adipose tissue compared to controls. The results show that glucose transport across the plasma membrane in vivo in most skeletal muscles tested, but not cardiac muscle, was increased by insulin administration to chicks. These findings suggest that an insulin-responsive glucose transport mechanism is present in chickens, even though they intrinsically lack GLUT4 homologous gene, the insulin-responsive glucose transporter in mammals.

Different effects of novel stressors on sympathoadrenal system activation in rats exposed to long-term immobilization.

Activation of the sympathoadrenal system, evaluated by plasma levels of epinephrine (E) and norepinephrine (NE) after exposure of rats to various stressors, is well documented. However, response of rats exposed long-term to a homotypic stressor and then exposed once to a heterotypic novel stressor is poorly understood. In the present study, we examined changes in plasma levels of catecholamines (CA) and corticosterone (COR) of rats after a single (2-h) or long-term repeated immobilization (41 times, 2 h daily) and in rats adapted to long-term immobilization exposed once to the novel stress of cold exposure or insulin or 2-deoxy-D-glucose (2DG) administration. Long-term immobilization produced a significant elevation of basal plasma COR but not NE and E levels. Long-term immobilized rats exposed to insulin or 2DG showed significant elevation of plasma CA and COR levels in comparison to the administration to control rats. Exposure of long-term immobilized and control rats to cold stress increased plasma NE and COR, whereas plasma E was not significantly changed. The exposure of long-term immobilized rats to a further single immobilization (2 h) increased plasma CA levels, but, in naive control rats, the single immobilization produced more pronounced increases. These data suggest that rats exposed to homotypic long-term immobilization are able to respond to heterotypic stressors by higher activation of the sympathoadrenal system as compared with the control, previously unstressed rats. Reduced plasma CA levels in long-term immobilized rats exposed to homotypic stressor are most probably due to an adaptation at the level of brain regulatory centers.