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Am J Physiol Endocrinol Metab 292: E1801-E1807, 2007. First published February 20, 2007; doi:10.1152/ajpendo.00700.2006
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Regulation of KATP channel subunit gene expression by hyperglycemia in the mediobasal hypothalamus of female rats

Maricedes Acosta-Martínez and Jon E. Levine

Department of Neurobiology and Physiology, Northwestern University, Evanston, Illinois

Submitted 20 December 2006 ; accepted in final form 13 February 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The ATP-sensitive potassium (KATP) channels are gated by intracellular adenine nucleotides coupling cell metabolism to membrane potential. Channels comprised of Kir6.2 and SUR1 subunits function in subpopulations of mediobasal hypothalamic (MBH) neurons as an essential component of a glucose-sensing mechanism in these cells, wherein uptake and metabolism of glucose leads to increase in intracellular ATP/ADP, closure of the channels, and increase in neuronal excitability. However, it is unknown whether glucose and/or insulin may also regulate the gene expression of the channel subunits in the brain. The present study investigated whether regulation of KATP channel subunit gene expression might be a mechanism by which neuronal populations adapt to prolonged changes in glucose and/or insulin levels in the periphery. Ovariectomized, steroid-replaced rats were fitted with indwelling jugular catheters and infused for 48 h with saline, glucose (hyperglycemia-hyperinsulinemia), insulin and glucose (hyperinsulinemia), diazoxide (control), or glucose and diazoxide (hyperglycemia). At the end of infusions, the MBH, preoptic area, and pituitary were dissected for RNA isolation and RT-PCR. Hyperglycemia decreased Kir6.2 mRNA levels in the MBH in both the presence and absence of hyperinsulinemia. These same conditions also produced a trend toward decreased SUR1 mRNA levels in the MBH; however, it did not exceed statistical significance. Hyperglycemia increased whereas hyperinsulinemia reduced neuropeptide Y mRNA levels when these groups were compared with each other. However, neither was significantly different from values observed in saline-infused controls. In conclusion, hyperglycemia per se may alter expression of KATP channels and thereby induce changes in the excitability of some MBH neurons.

adenosine triphosphate-sensitive potassium channels; glucose; insulin

THE ABILITY OF THE BRAIN to detect changes in peripheral metabolic signals is critical for the proper maintenance of energy homeostasis. Peripheral cues such as insulin, leptin, and glucose readily cross the blood-brain barrier (BBB) relaying information pertaining metabolic status (2, 16). This information is processed by specific brain centers which then trigger physiological and behavioral adjustments which are assumed to enhance individual survival and reproductive fitness (25). In recent years a number of mechanisms have been described to explain how the brain senses the metabolic status of the body. For example, specialized neurons in the brain respond to elevated exposure to glucose by altering their firing rate (12, 13). These "glucose-responsive neurons" (GR) increase their firing rate as extracellular glucose levels rise (4, 12, 13). The presence of ATP-sensitive potassium (KATP) channels is at the core of the capacity of these neurons to alter their membrane potential in response to changes in glucose (12, 13).

KATP channels are gated by intracellular adenine nucleotides and can thereby couple cell metabolic state to membrane potential and cell excitability (1). These channels are formed as heterooctameric protein complexes composed of four each of one of the indwardly rectifying K+ pore-forming subunits Kir6.1 or Kir6.2 and one of the sulfonylurea receptor (SUR) subunits SUR1 or SUR2A/B (1). In various neuronal populations, uptake and metabolism of glucose leads to an increase in the intracellular ATP/ADP ratio, closure of the channels, and a relative increase in neuronal excitability. Depending on the actual combination of pore-forming and sulfonylurea subunits, these channels show different affinities and responses to pharmacological agents. For example, KATP channels composed of the Kir6.2 and SUR1 are activated by diazoxide and blocked with high affinity by the sulfonylureas glybenclamide and tolbutamide (1).

Functional KATP channels have been identified in brain areas important for the control of food intake and energy balance, such as the arcuate nucleus of the hypothalamus (ARC) (38), the lateral hypothalamus (LH) (30), the paraventricular nucleus (11), and the ventromedial nucleus of the hypothalamus (VMH) (33). In these nuclei, the adipocyte hormone leptin and insulin, both known regulators of body weight (BW) and energy homeostasis, activate KATP channels on GR neurons (34, 35). In addition, electrophysiological and pharmacological studies have identified neuropeptide Y (NPY) and proopiomelanocortin (POMC) neurons as glucose responsive (4, 8). Both of these neuronal phenotypes and the neuropeptides they release are known potent regulators of feeding behavior (19, 27).

Although a direct role between KATP channel activity in GR neurons and the control of BW and energy homeostasis have not been established, several studies suggest some type of integrative role. Mice in which the pore-forming subunit of the KATP channel Kir6.2 has been deleted lack functional glucose-responsive neurons in the VMH (17). In the face of glucoprivic stimuli, these mice failed to show the humoral counterregulatory responses essential for the mobilization of glucose and the classical hyperphagic response that accompanies it (17). It has been shown that, in addition to adenosine nucleotides, membrane phospholipids such as phosphatidylinositol (PI) 3,4,5-triphosphate (PIP3) bind and modulate KATP channel activity (29). Recently, a POMC-specific inactivation of the gene for the PIP3 phosphatase PTEN resulted in marked hyperpolarization in these neurons due to increase in KATP channel activity (21). This POMC-specific disruption resulted in hyperphagia and a sexually dimorphic, diet-induced obesity. Furthermore, intraventricular administration of tolbutamide abolished the hyperphagia in these mice (21).

These and other studies have contributed to a better understanding about the mechanisms by which KATP channels are activated or inactivated depending on the cellular environment. The majority of these studies have focused on the effects of variations in glucose metabolism and on the actions of insulin and leptin signaling pathways such as PI3K, on neuronal firing rate. However, it is not known if dramatic changes in the plasma levels of these metabolic cues may also regulate the gene expression of the KATP channel subunits in the brain. This information would be of critical importance to shed light on how the brain adapts to prolong changes in glucose and or insulin such as those occurring during uncontrolled diabetes. Therefore, the present study investigated whether, in vivo, high levels of glucose and/or insulin affect KATP channel subunit mRNA levels in the female rat brain. Because the activity of POMC and NPY neurons is regulated by KATP channels and these peptides are critical for the regulation of both energy homeostasis and reproduction (25), we also investigated their specific transcriptional regulation under these conditions.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals and hormone treatments. To control for any influence of ovulatory cyclicity on gene expression, female rats (180–220 g) were bilaterally ovariectomized (OVX) and implanted with two Silastic capsules containing estrogen (E) and progesterone (P) (Sigma-Aldrich, St. Louis, MO). The hormone concentrations used have previously been shown to produce constant E and P levels of 20 pg/ml and 20 ng/ml, respectively, for at least 7 days. Three days after OVX, rats were fitted with an indwelling jugular vein catheter and allowed a 2-day period of recuperation (Fig. 1). The animals started in the weight range of 200–240 g and weighed 218 ± 0.22 g prior to the start of infusions (value expressed as mean ± SE). The Institutional Animal Care and Use Committee of Northwestern University approved this animal protocol.


Figure 1
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  		Fig. 1. Time course for surgeries and chronic infusions in female rats. OVX, ovariectomy.

 
Infusions. To manipulate glucose and insulin levels in vivo, we used the long-term infusion technique in unrestrained female rats (20). Three days after OVX, rats were fitted with a jugular catheter while they were under halothane anesthesia. Briefly, the right external jugular vein was exposed and temporarily occluded, and a small cut was made to accommodate insertion of a catheter consisting of a length of PE-50 tubing bearing a tip of flexible Silastic tubing (OD 1.19 mm). The Silastic tip was advanced to the atrium, and the tubing was secured to the vein and surrounding tissue. The remaining length of PE tubing was externalized via the nape and fitted with a stainless steel plug until subsequent access for infusions. A protective cuff served as an exit for the catheter from the subcutaneous space. Incisions were closed with silk sutures, and the animals were allowed to recover for 24 h before the beginning of the infusions (Fig. 1). Rats were randomly assigned to one of five experimental groups: 1) 0.9% NaCl-infused rats (controls), 2) diazoxide-infused rats (controls), 3) glucose-infused rats (high glucose-high insulin), 4) glucose- and diazoxide-infused rats (high glucose), and 5) insulin- and glucose-infused rats (high insulin). Infusions lasted for 48 h. A hypertonic (30% wt/vol) glucose solution was used while infusions of diazoxide were calculated on the basis of an infusion rate of 5 mg·kg–1·h–1. The flow rate was 50–70 µl/min, except for rats receiving a simultaneous infusion of insulin (30–50 mU/min), and glucose, in which case the glucose rate was adjusted to maintain euglycemia. To maintain the required ranges of glycemia and insulinemia during the infusion period, plasma glucose and insulin levels were measured five times daily in arteriovenous blood obtained from tail vessels by tail snipping. At the end of the infusion, trunk blood was collected, and the desired tissues, including the preoptic area (POA) and the MBH and pituitary were dissected and stored at –80°C until subjected to RNA extraction. The MBH was removed by inverting the brain and making transverse razor cuts at the caudal border of the optic chiasm and the rostral border of the mammilary bodies, medial to the lateral sulci and 1 mm deep. The POA was dissected as a block extending 2 mm rostrally from the caudal border of the optic chiasm, 2 mm dorsal to the ventral surface of the forebrain, and 1 mm to each side of the sagittal midline.

Analytic methods. Blood glucose was determined with a glucose analyzer (Prestige Smart System). The LH standard RP-3 and the antibody rLH-S11 were generously provided by National Institute of Diabetes and Digestive and Kidney Diseases. The sensitivity of the LH RIA was 0.2 ng/ml and the intra-assay coefficient of variance (CV) was 5.4%. Serum E2 concentrations were determined with RIA kits from Diagnostic Products (Los Angeles, CA). The sensitivity and intra-assay CV were 2 pg/ml and 4.1%, respectively. Serum insulin levels were determined using a rat RIA kit from Linco Research (St. Charles, MO). The sensitivity and intra-assay CV were 0.02 ng/ml and 7.4%, respectively.

Semiquantitative RT-PCR. Tissue was homogenized in TRIzol reagent (Invitrogen, Carlsbad CA) using a Polytron homogenizer (Brinkman Kinematra, Westbury, NY). Subsequently, RNA samples were treated with RQ RNase-free DNase (Promega, Madison, WI) and purified by phenol-chloroform extraction. Approximately 5 µg of total RNA were reversed transcribed into cDNA using 10x buffer (MgCl2 free; PerkinElmer), 10 mM dNTPs, 25 mM MgCl2, random hexamer primers, RNasin (33 U/µl, Promega), and Maloney murine leukemia virus reverse transcriptase (10 U/µl, Promega). The reaction mixture was incubated at 21°C for 10 min and held at 42°C for 75 min followed by 5 min at 95°C. The presence of contaminating DNA was tested by omitting reverse transcriptase in this step. Five microliters of each RT reaction were used for PCR reactions for the KATP channel subunits Kir6.2, SUR1, and SUR2A/B. The primers used were as follows: rat Kir6.2 (298-bp product, accession no. AB043638) sense (bases 497–516) 5'-gctgcatcttcatgaaaacg-3', reverse (bases 775–794) 5'-taccacgtcatcgactccaa-3'; rat SUR1 (400-bp product, accession no. NM_013039.1) sense (bases 2187–2206) 5'-tcaaggtcgtaaaccgcaag-3', reverse (bases 2567–2586) 5'-gcttacgcatctcagaaacc-3'; and rat SUR2A/B (501-bp product, accession no. D83598) sense (bases 1805–1823) 5'-GCAAGAGCGTGGAAGAGAC-3', reverse (bases 2305–2287) 5'-TGCCCCATGAGAAGTATCC-3'. Samples were normalized to the amplified product of the housekeeping gene RPL19 as internal control. PCR amplifications were performed in two steps as described previously (6, 36). In the first step, the reaction mixture contained 2 mM MgCl2, 0.2 mM of each of the four dNTPs, 12.5 pmol of each primer, 0.5 µCi [32P]CTP (Amersham, Arlington Heights, IL), Taq polymerase buffer, and 1.25 U Taq DNA polymerase (PerkinElmer, Norwalk, CT) in a final volume of 25 µl. After the appropriate number of cycles with the first set of primers, 20 µl of cocktail containing 1.25 U of additional Taq DNA polymerase were added in PCR buffer with 2 mM MgCl2 for the remaining cycles. After initial denaturation at 94°C for 4 min, the reaction mixture was subjected to successive cycles of denaturation at 94°C for 30 s, annealing at 55°C for 45 s, and extension at 72°C for 45 s, followed by extension at 70°C for 10 min after the final cycle. The optimal number of cycles, determined experimentally to yield a linear relationship between signal intensity and input cDNA, and an exponential one with respect to cycle number, was 32 in the case of KATP channel subunit PCR and 24 cycles with RPL19 primers. Radiolabeled PCR products were separated by electrophoresis in 6% nondenaturing polyacrylamide gels in Tris borate-EDTA buffer. Signal intensities were quantified by PhosphoImager with the Image Quant program (Molecular Dynamics, Sunnyvale, CA).

Real-time RT-PCR. A relative quantitation method was used to determine mRNA expression levels for NPY and POMC in the MBH. Primers for NPY, POMC, and GAPDH were obtained from published investigations (31). PCR was performed on a Bio-Rad iCycler using a 50-ng sample of hypothalamic cDNA added to iQ SYBR Green Supermix (Bio-Rad). NPY and POMC mRNA expressions were normalized to GAPDH, and a standard curve was used for each gene. mRNA content was expressed as a percentage of the mean value of saline-infused controls. Nontemplate controls were incorporated into each experiment.

Statistical analysis. Data are expressed as means ± SE. Statistical significance was determined with a one-way ANOVA test. The Newman-Keuls multiple comparison test was performed if significance was found. P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Plasma glucose and insulin concentrations during the infusions. Insulin and glucose levels during the 48-h infusions are reported in Fig. 2, A and B. As expected, infusion of glucose resulted in both hyperglycemia and hyperinsulinemia. Simultaneous infusion of glucose and diazoxide resulted in rats with constant hyperglycemia, while insulin levels were gradually decreased and reached close to normal levels between 8 and 12 h postinfusion. Coinfusion of insulin and glucose resulted in high plasma insulin levels, while close to normal (100–144 ng/ml) glucose levels were maintained throughout the infusion period. Finally, infusion of saline or diazoxide did not affect glucose or insulin levels.


Figure 2
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  		Fig. 2. Plasma insulin (A) and glucose (B) concentrations (ng/ml) during the 48-h infusion period in different treatment groups. Values presented are means ± SE; n = 6–7 female rats in each group.

 
At the end of the 48-h infusions, hyperleptinemia was observed in glucose-infused and glucose plus insulin-infused animals. However, leptin levels were significantly higher only in the latter group compared with the rest of the treatments (Table 1).


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  		Table 1. Plasma leptin concentration at the end of the 48-h infusions

 
Terminal steroid and gonadotropin concentrations. In these animals we controlled for variation in steroid levels by implanting Silastic capsules of E and P, so that all female rats were exposed to a similar ovarian steroid hormone milieu. E levels were not significantly different among the treatment groups (Table 2), confirming that the most important variables manipulated in these experiments were glucose, leptin, and insulin levels.


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  		Table 2. Plasma estrogen and LH levels measured at the end of the 48-h infusion

 
We also measured terminal plasma LH levels. There were no statistically significant differences in LH concentrations among the groups. However, a trend toward an increase in LH mean values in glucose- (high insulin-high glucose) and glucose plus diazoxide-infused (high glucose) groups was observed (Table 2).

Regulation of KATP channel subunit mRNA levels in the brain of female rats by hyperglycemia. We investigated Kir6.2, SUR1, and SUR2 mRNA levels in the MBH, POA, and pituitary of female rats in which insulin and/or glucose levels were chronically increased. In vivo infusions that induced high glucose levels resulted in a significant decrease in Kir6.2 mRNA levels in the MBH of female rats compared with diazoxide or saline-infused control groups (Fig. 3A). In contrast, when euglycemia was maintained while hyperinsulinemia was induced (glucose + insulin-infused group), Kir6.2 mRNA levels did not differ from control levels. SUR1 mRNA levels followed a similar trend but did not reach statistical significance (Fig. 3B). However, animals infused with glucose and insulin (high insulin-normal glucose) had a significantly higher SUR1 mRNA expression compared with the glucose- (high insulin-high glucose) and the glucose + diazoxide-infused (high glucose) group (Fig. 3B). There were no differences in SUR2 mRNA expression in the MBH among the groups. Therefore, in the MBH, glucose, but not insulin, decreases Kir6.2 mRNA expression.


Figure 3
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  		Fig. 3. Glucose, but not insulin, significantly decreased Kir6.2 mRNA subunit (A) gene expression in the mediobasal hypothalamus (MBH) of female rats, whereas a trend toward a decrease in SUR1 subunit (B) gene expression by glucose was observed. In contrast, hyperinsulinemia increased SUR1 subunit gene expression in this brain tissue compared with hyperglycemic rats. Values presented are means ± SE; n = 6–7. P = 0.002 and P = 0.017, ANOVA for Kir6.2 and SUR1, respectively. *P < 0.05, post hoc analysis compared with all other treatment groups. Bars marked with same letter are different from each other.

 
In vivo manipulations of glucose and insulin did not alter any of the KATP channel subunit mRNA levels in the POA of female rats (data not shown).

Finally, in the pituitary, Kir.6.2 and SUR1 mRNA levels were not significantly different among the groups (data not shown). However, SUR2A/B mRNA expression was decreased in rats infused with glucose and diazoxide (high glucose, Fig. 4). Post hoc analysis revealed that the levels in this group were significantly different from those in all the groups except the saline (control) group.


Figure 4
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  		Fig. 4. Hyperglycemia significantly reduced SUR2A/B mRNA levels in the pituitary of female rats. Values presented are means ± SE; n = 6–7. P = 0.0042, ANOVA. *P < 0.05, post hoc analysis compared with all other treatment groups.

 
Effect of hyperglycemia and/or hyperinsulinemia on hypothalamic NPY and POMC mRNA levels in female rats. As determined by real-time PCR, NPY mRNA content of the MBH in rats infused with glucose and diazoxide (hyperglycemia only) was increased compared with animals infused with insulin and glucose (hyperinsulemia only). The NPY mRNA levels in hyperglycemic/normoinsulinemic rats were also significantly higher than those in rats treated with diazoxide alone, although they were not significantly different from saline-infused control animals (Fig. 5). POMC mRNA content was not altered by any of the infusion paradigms in the MBH of female rats (data not shown).


Figure 5
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  		Fig. 5. Infusion of glucose and insulin (normal glucose, high insulin, and leptin levels) resulted in a significant decrease in neuropeptide Y (NPY) mRNA levels in the MBH of female rats. Values presented are means ± SE; n = ≥5, except glucose-infused group n = 3, P = 0.0337, ANOVA. Bars marked a are different from a, b different from b, *P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study is the first to demonstrate specific regulation of KATP channel subunit gene expression in the brain by a peripheral metabolic signal. Although a variety of in vivo models alter both glucose and insulin levels, our study differentiates between the effects of glucose and insulin on Kir6.2 and SUR1 channel subunit expression. We found that high glucose levels and not insulin significantly decreased Kir6.2 subunit mRNA levels in the MBH of female rats. Furthermore, we found that SUR2A/B mRNA expression was decreased in the pituitary of rats infused with glucose and diazoxide (high glucose). Finally, we observed a significant decrease in NPY mRNA levels under both hyperinsulinemic and hyperleptinemic conditions compared with hyperglycemia only.

Our results parallel in vitro and in vivo studies in which hyperglycemia induced a reduction in KATP channel gene expression in the pancreas. For example, cultured rat islets or the rat insulinoma cell line INS-I showed a significant decrease in Kir6.2 and SUR1 transcripts when exposed to high glucose concentrations (18). This was accompanied by a decrease in KATP channel activity. Conversely, glucose deprivation results in upregulation of the translation of existing Kir6.2 and SUR1 mRNAs (32). In rats, chronic (3 wk) hyperglycemia due to partial pancreatomy results in decreased KATP channel gene expression, which was associated with beta-cell hypertrophy and loss of beta-cell differentiation (9). Finally, male Zucker diabetic fatty rats showed a 34% decrease in expression of Kir6.2 mRNA in pancreatic islets compared with lean control rats (37). At least one study (14) has shown that streptozotocin-treated rats exhibited increased SUR binding in the hypothalamus, but the effect of hyperglycemia vs. the profound insulinopenia in these animals precludes attributing these effects to the hyperglycemic state. Indeed, the same group previously demonstrated that intracarotid infusions of glucose decreased SUR binding (15), consistent with the present findings.

In the ARC of the hypothalamus, NPY serves as a potent orexigenic neuropeptide (27). In this region, the expression of NPY is regulated by a number of peripheral signals, including glucose, leptin, and insulin (5, 23, 24, 26). We found that maintaining euglycemic levels while inducing high levels of insulin decreased NPY mRNA levels in the MBH of female rats compared with hyperglycemic animals. These results are in agreement with a number of in vivo studies in which central insulin administration is able to decrease NPY mRNA levels (5, 26). Moreover, in vivo models in which low levels of insulin are induced either pharmacologically or through fasting consistently result in high hypothalamic NPY levels and hyperphagia (5, 28). However, it is important to point out that we failed to see a similar response in the other infusion groups in which high levels of insulin and or glucose were induced. This discrepancy could be explained by a number of factors; for example, this infusion paradigm also resulted in significantly high levels of leptin. It is known that leptin can act centrally to decrease NPY expression in the hypothalamus (5). Acutely, glucose and insulin increase the transport of leptin into the brain of rodents (10). Therefore, the downregulation of NPY expression in this group could be the result not only of high insulin levels but of high leptin levels as well. Second, several lines of evidence point to brain region-specific effects of insulin or glucose on NPY gene expression (5, 26). It is possible that to see similar effects on the hyperglycemic and hyperinsulinemic groups a more discrete neuroanatomic approach such as in situ hybridization may be required. This could also explain the lack of effect on POMC gene expression by our treatments (data not shown).

KATP channels have been identified in the anterior pituitary of the rat and in pituitary cell lines (GH3 cells) (3). The K+ channel opener diazoxide decreased the basal level of growth hormone (GH) secretion (3). In the present study, a decrease in pituitary SUR2A/B mRNA levels by high plasma concentration of glucose was observed. This might result in altered GH levels such as those observed in diabetic rats and in normal rats with hypoglycemia, where GH levels are suppressed.

Transcriptional control of neuropeptide gene expression is an important mechanism for the regulation of orexigenic and anorexigenic pathways in the hypothalamus. However, recent evidence suggests that the regulation of neuronal excitability represents an additional mechanism that could determine the release and, hence, physiological outcome of these neuroregulators. For example, POMC neurons are glucose responsive and express KATP channels (8). By use of the CRE recombinase genetic manipulation, it was recently demonstrated that the specific constitutive activation of PIP3 formation in POMC cells caused KATP channel activation (21). This, in turn, results in hyperphagia, diet-sensitive obesity, and leptin resistance in these mice. The authors concluded that augmented PIP3-dependent KATP channel activation results in electrical silencing of POMC neurons. Given that both NPY and POMC neurons express KATP channels, suppression of KATP channel gene expression by high levels of glucose could potentially increase the excitability of both types of neurons. A decrease in KATP channel expression and function by glucose could inform the brain that fuel is present and, hence, indicate satiety. However, the net effect on feeding behavior and energy homeostasis likely depends on the combined influence of glucose and other peripheral signals, such as insulin and leptin.

A reduction in brain KATP channel gene expression could also have implications for the control of peripheral glucose homeostasis. Intracerebroventricular infusion of insulin diminished hepatic glucose output, and blockade of MBH KATP channels with the sulfonylurea tolbutamide abolished this effect (22). Therefore, a decrease in central KATP channel function might affect the ability of insulin signaling to control glucose homeostasis in the periphery.

It is well documented that conditions of metabolic imbalance or impaired glucose homeostasis result in a parallel disruption of hypothalamic KATP channel activity (4). For example, in rodent models of diet-induced obesity and type 2 diabetes, there is a decrease in the number of glucosensing neurons, and those remaining glucose responsive showed abnormal responses to glucose. In addition, the ability of physiological levels of insulin or leptin to activate GR neuron KATP channels in the VMH is absent in genetically obese Zucker rats (fa/fa) (34, 35). Although we did not measure neuronal KATP channel activity after our infusion treatments, on the basis of these studies we could speculate that the decrease in Kir6.2 gene expression under hyperglycemia is accompanied by a decrease in KATP channel activity.

The infusion paradigms employed in the present study most likely mimicked, albeit for a relatively short time, circumstances in uncontrolled diabetes where supraphysiological plasma glucose and insulin levels are observed (7). Our data suggest that alterations in the gene expression of both orexigenic/anorexigenic neuropeptides and of KATP channel subunits can contribute to the metabolic imbalance observed during such conditions. In humans, uncontrolled diabetes adversely affects brain metabolism and cognitive function (7, 16). The notion that glucose per se regulates channel gene expression provides an important mechanism by which long-term alterations in neuronal function can occur.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Institute of Child Health and Human Development Grants RO1-HD-20677, P50-HD-44405, and T32-HD-07068.


   ACKNOWLEDGMENTS
 
We thank Felix Nuñez, Rada Rajasingham, Adriane Sinclaire, and Katie Dorton for technical support and Brigitte Mann for technical expertise in performing RIAs.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. E. Levine, Dept. of Neurobiology and Physiology, Northwestern University, 2205 Tech Dr., Evanston, IL 60208 (e-mail: jlevine{at}northwestern.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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