Changes In Extracellular Glucose Research Articles

Reduction in mitochondrial ATP synthesis mimics the effect of low glucose in depolarizing neurons from the subpostremal nucleus of the solitary tract of rats.

Neurons of the subpostremal nucleus of the solitary tract (NTS) respond to changes in extracellular glucose with alterations in membrane potential with both depolarization and hyperpolarization. From 5 mM glucose, a rapid shift to 0.5 mM glucose produces a membrane depolarization by an unknown mechanism in most neurons. However, the mechanism involved in this response needs to be known. Here, we investigated if the low glucose-induced depolarization could be mimicked by reducing ATP synthesis and possible mediators of this effect. We showed that applying the mitochondrial uncoupler CCCP (1 µM) reproduced the effects of low glucose depolarizing the membrane, generating an inward current, and decreasing membrane resistance. On the other hand, activation of AMPK did not alter these parameters. To test if low glucose and CCCP could depolarize the membrane by affecting the ionic gradient, we inhibited the electrogenic Na/K pump with 10 µM of ouabain. We observed a similar membrane depolarization but not a decrease in membrane resistance. We conclude that perfusion of neurons of the subpostremal NTS with a low glucose solution depolarizes the membrane by probably reducing intracellular ATP, but not by activating AMPK or decreasing the ionic gradient across the membrane.

The impact of glucose exposure on bioenergetics and function in a cultured endothelial cell model and the implications for cardiovascular health in diabetes

Cardiovascular disease is the primary driver of morbidity and mortality associated with diabetes. Hyperglycaemia is implicated in driving endothelial dysfunction that might underpin the link between diabetes and cardiovascular disease. This study was designed to determine the impact of chronic preconditioning of cells to hyperglycaemia and transient switching of cultured endothelial cells between hyper- and normo-glycaemic conditions on bioenergetic and functional parameters. Immortalised EA.hy926 endothelial cells were cultured through multiple passages under normoglycaemic (5.5 mM) or hyperglycaemic (25 mM) conditions. Cells were subsequently subjected (48 h) to continued normo- or hyperglycaemic exposure, or were switched to the alternative glycaemic condition, or to an intermediate glucose concentration (12.5 mM) and metabolic activity, together with key markers of function were measured. Cells habituated to hyperglycaemia were energetically quiescent. Functional activity, characterised by the measurement of nitric oxide, endothelin-1, tissue plasminogen activator and plasminogen activator inhibitor-1, was depressed by exposure to high glucose, with the reduction in nitric oxide production being the most notable. Function was more responsive to acute changes in extracellular glucose than were bioenergetic changes. We conclude that glucose is a key determinant of endothelial function. The study highlights the importance of chronic glucose exposure on cell phenotype and emphasises the need to pay close attention to glucose preconditioning in interpreting results under culture conditions.

Evaluation of Anti-proliferative Effects of Barringtonia racemosa and Gallic Acid on Caco-2 Cells

Barringtonia racemosa leaf water extract (BLE) had been shown to have high gallic acid (GA) content and BLE has been postulated to have anti-proliferative effects towards colorectal cancer. This study aims to further investigate the mechanism underlying the anti-proliferative effect of BLE in Caco-2 cells and to determine if GA is responsible for the observed effects. Both BLE and GA inhibited Caco-2 cells in a dose-dependent manner. Cells exposed to IC50 concentration of BLE and GA showed reduced antioxidant activities. GA-treated Caco-2 cells experienced higher oxidative stress compared to cells treated with BLE. Both BLE and GA significantly up-regulated the expression of SLC2A1. BLE but not GA, significantly down-regulated the expression of ADH4. Meanwhile, GA but not BLE, significantly up-regulated AKRIB10 and GLO1 but significantly down-regulated HAGH. Alterations in gene expression were coupled with changes in extracellular glucose and pyruvate levels. While BLE decreased intracellular pyruvate, GA did the opposite. Both intracellular and extracellular D-lactate were not affected by either BLE or GA. GA showed more pronounced effects on apoptosis while BLE irreversibly reduced cell percentage in the G0/G1 phase. In conclusion, this study demonstrates the multiple-actions of BLE against Caco-2 cells, potentially involving various polyphenolic compounds, including GA.

Changes in neuronal activity across the mouse ventromedial nucleus of the hypothalamus in response to low glucose: Evaluation using an extracellular multi-electrode array approach.

The hypothalamic ventromedial nucleus (VMN) is involved in maintaining systemic glucose homeostasis. Neurophysiological studies in rodent brain slices have identified populations of VMN glucose‐sensing neurones: glucose‐excited (GE) neurones, cells which increased their firing rate in response to increases in glucose concentration, and glucose‐inhibited (GI) neurones, which show a reduced firing frequency in response to increasing glucose concentrations. To date, most slice electrophysiological studies characterising VMN glucose‐sensing neurones in rodents have utilised the patch clamp technique. Multi‐electrode arrays (MEAs) are a state‐of‐the‐art electrophysiological tool enabling the electrical activity of many cells to be recorded across multiple electrode sites (channels) simultaneously. We used a perforated MEA (pMEA) system to evaluate electrical activity changes across the dorsal‐ventral extent of the mouse VMN region in response to alterations in glucose concentration. Because intrinsic (ie, direct postsynaptic sensing) and extrinsic (ie, presynaptically modulated) glucosensation were not discriminated, we use the terminology ‘GE/presynaptically excited by an increase (PER)’ and ‘GI/presynaptically excited by a decrease (PED)’ in the present study to describe responsiveness to changes in extracellular glucose across the mouse VMN. We observed that 15%‐60% of channels were GE/PER, whereas 2%‐7% were GI/PED channels. Within the dorsomedial portion of the VMN (DM‐VMN), significantly more channels were GE/PER compared to the ventrolateral portion of the VMN (VL‐VMN). However, GE/PER channels within the VL‐VMN showed a significantly higher basal firing rate in 2.5 mmol l‐1 glucose than DM‐VMN GE/PER channels. No significant difference in the distribution of GI/PED channels was observed between the VMN subregions. The results of the present study demonstrate the utility of the pMEA approach for evaluating glucose responsivity across the mouse VMN. pMEA studies could be used to refine our understanding of other neuroendocrine systems by examining population level changes in electrical activity across brain nuclei, thus providing key functional neuroanatomical information to complement and inform the design of single‐cell neurophysiological studies.

Platinized carbon fiber-based glucose microbiosensor designed for metabolic studies in brain slices

In order to understand how energy metabolism adapts to changes in neuronal activity it is imperative to perform direct measurements of the flux of glucose (and other metabolites) in brain tissue. Metabolic studies using brain slice preparations are attractive due to the controllability of recording conditions, absence of anesthetic interference and refined animal experimental protocols. In this work, taking advantage of the small size and versatility of carbon fiber microelectrodes (CFMs), we aimed to develop an amperometric glucose microbiosensor suitable for glucose measurement in brain slices. Potentiostatic- or galvanostatic-driven platinum electrodeposition was used to improve the analytical properties of CFMs towards detection of hydrogen peroxide. The platinized CFMs served as platform for the development of glucose microbiosensors through the immobilization of glucose-oxidase (GOx) by cross-linking with glutaraldehyde in the presence of BSA. Selective glucose measurements were attained by modifying the electrode with a permselective layer of meta-phenylenediamine and by integrating a null sensor. The in vitro characterization studies support the good analytical features of the CFM/Pt-based microbiosensors to reliably measure glucose in brain tissue. The ex vivo experiments in rodent hippocampal slices validated their suitability to measure evoked changes in extracellular glucose. This approach, encompassing the use of null sensor to cross-check the selectivity on a moment-to-moment basis, allowed us to provide the temporal and quantitative profile of extracellular glucose changes in hippocampal slices following a spreading depolarization event.Overall, these results support the potential of these microbiosensors to be used as a valuable tool to investigate the complex nature of glucose utilization in brain tissue linked to neuronal activation both in physiological and pathological conditions.

Modelling the impact of changes in the extracellular environment on the cytosolic free NAD+/NADH ratio during cell culture.

Cancer cells depend on glucose metabolism via glycolysis as a primary energy source, despite the presence of oxygen and fully functioning mitochondria, in order to promote growth, proliferation and longevity. Glycolysis relies upon NAD+ to accept electrons in the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) reaction, linking the redox state of the cytosolic NAD+ pool to glycolytic rate. The free cytosolic NAD+/NADH ratio is involved in over 700 oxidoreductive enzymatic reactions and as such, the NAD+/NADH ratio is regarded as a metabolic readout of overall cellular redox state. Many experimental techniques that monitor or measure total NAD+ and NADH are unable to distinguish between protein-bound and unbound forms. Yet total NAD+/NADH measurements yield little information, since it is the free forms of NAD+ and NADH that determine the kinetic and thermodynamic influence of redox potential on glycolytic rate. Indirect estimations of free NAD+/NADH are based on the lactate/pyruvate (L/P) ratio at chemical equilibrium, but these measurements are often undermined by high lability. To elucidate the sensitivity of the free NAD+/NADH ratio to changes in extracellular substrate, an in silico model of hepatocarcinoma glycolysis was constructed and validated against in vitro data. Model simulations reveal that over experimentally relevant concentrations, changes in extracellular glucose and lactate concentration during routine cancer cell culture can lead to significant deviations in the NAD+/NADH ratio. Based on the principles of chemical equilibrium, the model provides a platform from which experimentally challenging situations may be examined, suggesting that extracellular substrates play an important role in cellular redox and bioenergetic homeostasis.

Fluctuations of extracellular glucose and lactate in the mouse primary visual cortex during visual stimulation

We measured the extracellular glucose and lactate in the primary visual cortex in the CD-1 mouse using electrochemical electrodes. To gain some additional information on brain metabolism, we examined the impact of systemic injections of lactate and fructose on the brain extracellular glucose and lactate changes observed during visual stimulation. We found that simple stimulation using a flashlight produced a decrease in visual cortex extracellular glucose and an increase in extracellular lactate. Similar results were observed following visual stimulation with an animated movie without soundtrack or the presentation of a novel object. Specificity of these observations was confirmed by the absence of extracellular glucose and lactate changes when the mice were presented a second time with the same object. Previous experiments have shown that systemic injections of fructose and lactate lead to an increase in blood lactate but no change in blood glucose while they both increase brain extracellular glucose but they do not increase brain extracellular lactate. When mice were visually stimulated after they had received these injections, we found that lactate, and to a slightly lesser degree fructose, both reduced the amplitude of the changes in extracellular glucose and lactate that accompanied visual stimulation. Thus, neural activation leads to an increase in extracellular lactate and a decrease in extracellular glucose. Novelty, attentional resources and availability of metabolic fuels modulate these fluctuations. The observations are consistent with a modified view of brain metabolism that takes into account the blood and brain glucose availability.

Glucose-Sensing in the Reward System.

Glucose-sensing neurons are neurons that alter their activity in response to changes in extracellular glucose. These neurons, which are an important mechanism the brain uses to monitor changes in glycaemia, are present in the hypothalamus, where they have been thoroughly investigated. Recently, glucose-sensing neurons have also been identified in brain nuclei which are part of the reward system. However, little is known about the molecular mechanisms by which they function, and their role in the reward system. We therefore aim to provide an overview of molecular mechanisms that have been studied in the hypothalamic glucose-sensing neurons, and investigate which of these transporters, enzymes and channels are present in the reward system. Furthermore, we speculate about the role of glucose-sensing neurons in the reward system.

Acute Effects of Sugars and Artificial Sweeteners on Small Intestinal Sugar Transport: A Study Using CaCo-2 Cells As an In Vitro Model of the Human Enterocyte.

BackgroundThe gastrointestinal tract is responsible for the assimilation of nutrients and plays a key role in the regulation of nutrient metabolism and energy balance. The molecular mechanisms by which intestinal sugar transport are regulated are controversial. Based on rodent studies, two models currently exist that involve activation of the sweet-taste receptor, T1R2/3: an indirect model, whereby luminal carbohydrates activate T1R2/3 expressed on enteroendocrine cells, resulting in the release of gut peptides which in turn regulate enterocyte sugar transport capacity; and a direct model, whereby T1R2/3 expressed on the enterocyte regulates enterocyte function.AimsTo study the direct model of intestinal sugar transport using CaCo-2 cells, a well-established in vitro model of the human enterocyte.MethodsUptake of 10mM 14C D-Glucose and D-Fructose into confluent CaCo-2/TC7 cells was assessed following 3hr preincubation with sugars and artificial sweeteners in the presence and absence of the sweet taste receptor inhibitor, lactisole. Expression of the intestinal sugar transporters and sweet-taste receptors were also determined by RT-PCR.ResultsIn response to short term changes in extracellular glucose and glucose/fructose concentrations (2.5mM to 75mM) carrier-mediated sugar uptake mediated by SGLT1 and/or the facilitative hexose transporters (GLUT1,2,3 and 5) was increased. Lactisole and artificial sweeteners had no effect on sugar transport regulated by glucose alone; however, lactisole increased glucose transport in cells exposed to glucose/fructose. RT-PCR revealed Tas1r3 and SGLT3 gene expression in CaCo-2/TC7 cells, but not Tas1r2.ConclusionsIn the short term, enterocyte sugar transport activities respond directly to extracellular glucose levels, but not fructose or artificial sweeteners. We found no evidence of a functional heterodimeric sweet taste receptor, T1R2/3 in CaCo-2 cells. However, when glucose/fructose is administered together there is an inhibitory effect on glucose transport possibly mediated by T1R3.

Lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNFα) blunt the response of Neuropeptide Y/Agouti-related peptide (NPY/AgRP) glucose inhibited (GI) neurons to decreased glucose

A population of Neuropeptide Y (NPY) neurons which co-express Agouti-related peptide (AgRP) in the arcuate nucleus of the hypothalamus (ARC) are inhibited at physiological levels of brain glucose and activated when glucose levels decline (e.g. glucose-inhibited or GI neurons). Fasting enhances the activation of NPY/AgRP-GI neurons by low glucose. In the present study we tested the hypothesis that lipopolysaccharide (LPS) inhibits the enhanced activation of NPY/AgRP-GI neurons by low glucose following a fast. Mice which express green fluorescent protein (GFP) on their NPY promoter were used to identify NPY/AgRP neurons. Fasting for 24h and LPS injection decreased blood glucose levels. As we have found previously, fasting increased c-fos expression in NPY/AgRP neurons and increased the activation of NPY/AgRP-GI neurons by decreased glucose. As we predicted, LPS blunted these effects of fasting at the 24h time point. Moreover, the inflammatory cytokine tumor necrosis factor alpha (TNFα) blocked the activation of NPY/AgRP-GI neurons by decreased glucose. These data suggest that LPS and TNFα may alter glucose and energy homeostasis, in part, due to changes in the glucose sensitivity of NPY/AgRP neurons. Interestingly, our findings also suggest that NPY/AgRP-GI neurons use a distinct mechanism to sense changes in extracellular glucose as compared to our previous studies of GI neurons in the adjacent ventromedial hypothalamic nucleus.

Behavior-associated and post-consumption glucose entry into the nucleus accumbens extracellular space during glucose free-drinking in trained rats.

Glucose is the primary energetic substrate for the metabolic activity of brain cells and its proper delivery from the arterial blood is essential for neural activity and normal brain functions. Glucose is also a unique natural reinforcer, supporting glucose-drinking behavior without food or water deprivation. While it is known that glucose enters brain tissue via gradient-dependent facilitated diffusion, it remains unclear how glucose levels are changed during natural behavior and whether the direct central action of ingested glucose can be involved in regulating glucose-drinking behavior. Here, we used glucose biosensors with high-speed amperometry to examine the pattern of phasic and tonic changes in extracellular glucose in the nucleus accumbens (NAc) during unrestricted glucose-drinking in well-trained rats. We found that the drinking behavior is highly cyclic and is associated with relatively large and prolonged increases in extracellular glucose levels. These increases had two distinct components: a highly phasic but relatively small behavior-related rise and a larger tonic elevation that results from the arrival of consumed glucose into the brain’s extracellular space. The large post-ingestion increases in NAc glucose began minutes after the cessation of drinking and were consistently associated with periods of non-drinking, suggesting that the central action of ingested glucose could inhibit drinking behavior by inducing a pause in activity between repeated drinking bouts. Finally, the difference in NAc glucose responses found between active, behavior-mediated and passive glucose delivery via an intra-gastric catheter confirms that motivated behavior is also associated with metabolic glucose use by brain cells.

Glycemic State Regulates Brain Derived Neurotrophic Factor Responsiveness of Neurons in the Paraventrucular Nucleus

Brain derived neurotrophic factor (BDNF) is a chemical messenger that has been implicated in the central control of food intake and regulation of the stress axis. Consistent with a role in feeding behavior, TrkB, the receptor through which BDNF exerts its effects, is expressed in the paraventricular nucleus (PVN). The PVN is a complex integrative autonomic centre with critical roles in the coordinated control of food intake. Recently, studies have shown that glucose‐sensing neurons in areas of the hypothalamus, including PVN, exhibit altered excitability in response to changes in extracellular glucose. Here, whole cell current‐clamp recordings were employed on rat PVN neurons in slice preparation to evaluate the ability of these cells to integrate multiple metabolically relevant signals. BDNF (2nM) was bath applied in both high (10mM) and low (0.2mM) extracellular glucose concentrations. BDNF applied to PVN neurons (N=48) in 10mM glucose elicited varying responses with 50% depolarizing (mean change in membrane potential ± standard error: 9.0 ± 1.2 mV), 23% hyperpolarizing (mean: ‐6.7 ± 1.4 mV), and 27% showing no response to BDNF treatment. However, BDNF's effects were profoundly altered in hypoglycemic conditions. BDNF application to PVN neurons in 0.2mM glucose (N=15) elicited primarily hyperpolarizing effects (73%, mean: ‐6.4 ± 0.9 mV) apart from one cell that depolarized (7%, mean: 4.8 mV). Our findings demonstrate the potential role of BDNF in feeding through its impact on the excitability of PVN neurons and how these responses are altered by glycemic state.

D-Alanine in the islets of Langerhans of rat pancreas

Relatively high levels of d-alanine (d-Ala), an endogenous d-amino acid, have been found in the endocrine systems of several animals, especially in the anterior pituitary; however, its functional importance remains largely unknown. We observed d-Ala in islets of Langerhans isolated from rat pancreas in significantly higher levels than in the anterior/intermediate pituitary; specifically, 180±60fmol d-Ala per islet (300±100nmol/gislet), and 10±2.5nmol/g of wet tissue in pituitary. Additionally, 12±5% of the free Ala in the islets was in the d form, almost an order of magnitude higher than the percentage of d-Ala found in the pituitary. Surprisingly, glucose stimulation of the islets resulted in d-Ala release of 0.6±0.5fmol per islet. As d-Ala is stored in islets and released in response to changes in extracellular glucose, d-Ala may have a hormonal role.

Membrane potential dye imaging of ventromedial hypothalamus neurons from adult mice to study glucose sensing.

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with increasing connective tissue and decreased neuronal viability that occur with age. Here, we describe a methodology for the dissociation of healthy hypothalamic neurons from adult-aged mice. The ability to study neurons from adult-aged mice allows the use of disease models that manifest at a later age and might be more developmentally accurate for certain studies. Fluorescence imaging of dissociated neurons can be used to study the activity of a population of neurons, as opposed to using electrophysiology to study a single neuron. This is particularly useful when studying a heterogeneous neuronal population in which the desired neuronal type is rare such as for hypothalamic glucose sensing neurons. We utilized membrane potential dye imaging of adult ventromedial hypothalamic neurons to study their responses to changes in extracellular glucose. Glucose sensing neurons are believed to play a role in central regulation of energy balance. The ability to study glucose sensing in adult rodents is particularly useful since the predominance of diseases related to dysfunctional energy balance (e.g. obesity) increase with age.

Regulation of glucagon secretion by glucose: paracrine, intrinsic or both?

Glucagon secretion is regulated by glucose but the mechanisms involved remain hotly debated. Both intrinsic (within the α-cell itself) and paracrine (mediated by factors released β- and/or δ-cells) have been postulated. Glucagon secretion is maximally suppressed by glucose concentrations that do not affect insulin and somatostatin secretion, a finding that highlights the significance of intrinsic regulation of glucagon secretion. Experiments on islets from mice lacking functional ATP-sensitive potassium channels (K(ATP)-channels) indicate that these channels are critical to the α-cell's capacity to sense changes in extracellular glucose. Here, we review recent data on the intrinsic and paracrine regulation of glucagon secretion in human pancreatic islets. We propose that glucose-induced closure of the K(ATP)-channels, via membrane depolarization, culminates in reduced electrical activity and glucagon secretion by voltage-dependent inactivation of the ion channels involved in action potential firing. We further demonstrate that glucagon secretion measured in islets isolated from donors with type-2 diabetes is reduced at low glucose and that glucose stimulates rather than inhibits secretion in these islets. We finally discuss the relative significance of paracrine and intrinsic regulation in the fed and fasted states and propose a unifying model for the regulation of glucagon secretion that incorporates both modes of control.

Glucosensing by GnRH Neurons: Inhibition by Androgens and Involvement of AMP-Activated Protein Kinase

GnRH neurons integrate steroidal and metabolic cues to regulate fertility centrally. Central glucoprivation reduces LH secretion, which is governed by GnRH release, suggesting GnRH neuron activity is modulated by glucose availability. Here we tested whether GnRH neurons can sense changes in extracellular glucose, and whether glucosensing is altered by the steroids dihydrotestosterone (DHT) and/or estradiol (E). Extracellular recordings were made from GnRH neurons in brain slices from ovariectomized (OVX) mice ± DHT and/or E implants. Firing rate was reduced by a switch from 4.5 to 0.2 mm glucose in cells from OVX, OVX+E, and OVX+DHT+E mice, but not OVX+DHT mice. This suggests that androgens reduce the sensitivity of GnRH neurons to changes in extracellular glucose, but E mitigates this effect. Next we investigated potential mechanisms. In the presence of the ATP-sensitive potassium channel antagonist tolbutamide, glucosensing persisted. In contrast, glucosensing was attenuated in the presence of compound C, an antagonist of AMP-activated protein kinase (AMPK), suggesting a role for AMPK in glucosensing. The AMPK activator N1-(b-D-ribofuranosyl)-5-aminoimidazole-4-carboxamide (AICAR) mimicked the effect of low glucose and was less effective in cells from DHT-treated mice. The effect of DHT to diminish responses to low glucose and AICAR was abolished by blockade of fast synaptic transmission. Both AICAR and low glucose activated a current with a reversal potential near -50 mV, suggesting a nonspecific cation current. These studies indicate that glucosensing is one mechanism by which GnRH neurons sense fuel availability and point to a novel role for AMPK in the central regulation of fertility.

Polysaccharide Multilayer Nanoencapsulation of Insulin-Producing β-Cells Grown as Pseudoislets for Potential Cellular Delivery of Insulin

This paper describes the use of a layer-by-layer nanocoating technique for the encapsulation of insulin-producing pancreatic beta-cell spheroids (pseudoislets) within chitosan/alginate multilayers. We used pseudoislets self-organized from a population of the insulinoma cell line MIN6, derived from a transgenic mouse expressing the large T-antigen of SV40 in pancreatic beta-cells, as an experimental model for the study of cell nanoencapsulation. The maintenance of spheroid morphology and retention of cell viability and metabolic functionality was demonstrated postencapsulation. By depositing an additional protein-repelling phosphorylcholine-modified chondroitin-4-sulfate layer, the coatings were found to shield effectively access of large molecules of the immune systems to the antigen-presenting cell surfaces. Transmission electron microscopy analysis of the encapsulated pseudoislets revealed that the coating did not damage the cell structure. In addition, nanoencapsulation permits the cells to respond to changes in extracellular glucose and other insulin secretagogues by releasing insulin with a profile similar to that of nonencapsulated cells. These results suggest that this nanofilm encapsulation technique has the characteristics required for the efficient transplantation of cellular engineered beta-cells as a cell replacement therapy for type 1 diabetes. This encapsulation method is general in scope and has implications for use in a variety of cellular therapeutics employing engineered tissues from cells generated in vitro from various sources, including those using genetic and cellular engineering techniques.

Neuroendocrine, metabolic and pharmacological control of feeding behaviour – closing in on antiobesity treatment

Neuroendocrine, metabolic and pharmacological control of feeding behaviour – closing in on antiobesity treatment

Hyperglycemia impairs glucose and insulin regulation of nitric oxide production in glucose-inhibited neurons in the ventromedial hypothalamus

Physiological changes in extracellular glucose, insulin, and leptin regulate glucose-excited (GE) and glucose-inhibited (GI) neurons in the ventromedial hypothalamus (VMH). Nitric oxide (NO) signaling, which is involved in the regulation of food intake and insulin signaling, is altered in obesity and diabetes. We previously showed that glucose and leptin inhibit NO production via the AMP-activated protein kinase (AMPK) pathway, while insulin stimulates NO production via the phosphatidylinositol-3-OH kinase (PI3K) pathway in VMH GI neurons. Hyperglycemia-induced inhibition of AMPK reduces PI3K signaling by activating the mammalian target of rapamycin (mTOR). We hypothesize that hyperglycemia impairs glucose and insulin-regulated NO production in VMH GI neurons. This hypothesis was tested in VMH neurons cultured in hyperglycemic conditions or from streptozotocin-induced type 1 diabetic rats using NO- and membrane potential-sensitive dyes. Neither decreased extracellular glucose from 2.5 to 0.5 mM, nor 5 nM insulin increased NO production in VMH neurons in either experimental condition. Glucose- and insulin-regulated NO production was restored in the presence of the AMPK activator, 5-aminoimidazole-4-carboxamide-1-b-4-ribofuranoside or the mTOR inhibitor rapamycin. Finally, decreased glucose and insulin did not alter membrane potential in VMH neurons cultured in hyperglycemic conditions or from streptozotocin-induced rats. These data suggest that hyperglycemia impairs glucose and insulin regulation of NO production through AMPK inhibition. Furthermore, glucose and insulin signaling pathways interact via the mTOR pathway.

Regulation of Pancreas Duodenum Homeobox-1 Expression by Early Growth Response-1

The homeodomain transcription factor pancreas duodenum homeobox-1 (PDX-1) is a key regulator of pancreatic beta-cell development, function, and survival. Deficits in PDX-1 expression result in insulin deficiency and hyperglycemia. We previously found that the glucose-responsive transcription factor early growth response-1 (Egr-1) activates the insulin promoter in part by increasing expression levels of PDX-1. We now report that Egr-1 binds and activates multiple regulatory sites within the pdx-1 promoter. We identified consensus Egr-1 recognition sequences within proximal and distal regions of the mouse pdx-1 promoter and demonstrated specific binding of Egr-1 by chromatin immunoprecipitation and electrophoretic mobility shift assays. Overexpression of Egr-1 increased transcriptional activation of the -4500 proximal pdx-1 promoter and of the highly conserved regulatory Areas I, II, and III. Mutagenesis of a specific Egr-1 binding site within Area III substantially decreased Egr-1-mediated activation. Egr-1 increased the transcriptional activation of Areas I and II, despite the absence of Egr-1 recognition sequences within this promoter segment, suggesting that Egr-1 also can regulate the pdx-1 promoter indirectly. Egr-1 increased, and a dominant-negative Egr-1 mutant repressed, the transcriptional activation of distal pdx-1 promoter sequences. Mutagenesis of a specific Egr-1 binding site within regulatory Area IV reduced basal and Egr-1-mediated transcriptional activation. Our data indicate that Egr-1 regulates expression of PDX-1 in pancreatic beta-cells by both direct and indirect activation of the pdx-1 promoter. We propose that Egr-1 expression levels may act as a sensor in pancreatic beta-cells to translate extracellular signals into changes in PDX-1 expression levels and pancreatic beta-cell function.