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Effect of Diazoxide on Brain Capillary Insulin Receptor Binding and Food Intake in Hyperphagic Obese Zucker Rats¹

Department of Pediatrics, University of Tennessee Medical Center, Knoxville, Tennessee 37920

Address all correspondence and requests for reprints to: Ramin Alemzadeh, MD, Department of Pediatrics, University of Tennessee Medical Center, 1924 Alcoa Highway, U-1 13, Knoxville, Tennessee 37920. E-mail: ralemzad{at}mc.utinck.edu

	Abstract

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Insulin is believed to act as a central adiposity signal by binding to hypothalamic and other brain insulin receptors. Entry of circulating insulin into the brain is accomplished by a saturable receptor-mediated transendothelial transport system and is believed to be impaired in hyperinsulinemic, insulin-resistant, and hyperphagic obese Zucker rats. Theoretically, if hyperinsulinemia is decreased simultaneously while brain capillary insulin binding is increased, uptake of insulin into the brain can be enhanced leading to reduced food intake. To test this hypothesis, we administered diazoxide (DZ, 150 mg/kg/day), an inhibitor of glucose-mediated insulin secretion, or vehicle (control) to 7-week-old female obese and lean Zucker rats for 4 weeks (n = 24–28/subgroup-strain). Animals were assigned to either fasted (FD) or free-fed (FF) protocol for determination of plasma and cerebrospinal fluid (CSF) insulin and brain capillary insulin binding at the end of 4 weeks. DZ obese consumed fewer calories (P < 0.01) and gained less weight than control obese (P < 0.01), whereas DZ lean had similar amounts of caloric intake and weight gain compared with lean controls. DZ obese had lower fasting and random plasma glucose than control obese (P < 0.05). FD and FF DZ-treated obese and lean rats had lower plasma insulin than their respective obese (P < 0.01) and lean (P < 0.01) controls. FD and FF DZ-treated obese rats demonstrated higher CSF insulin (P < 0.05) and CSF/plasma insulin ratio (P < 0.01) than their controls, while only FF DZ lean animals showed higher CSF/plasma insulin ratio (P < 0.01) than their controls (P < 0.05). This was associated with enhanced brain capillary insulin binding in FD and FF DZ-treated obese (P < 0.01) and lean (P < 0.05) animals compared with their respective controls. It was concluded that DZ treatment in obese Zucker rats caused a decrease in insulin secretion and partially reversed impaired insulin binding to brain capillaries, leading to enhanced brain insulin uptake, and resulted in reduced food intake and weight gain observed in these animals.

	Introduction

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IT has been previously proposed that cerebrospinal fluid (CSF) insulin provides a feedback signal to the central nervous system (CNS) for suppression of food intake (1, 2). Infusion of insulin into the hypothalamic or ventricular CSF produces satiety and weight loss (3, 4), and insulin is suggested to be a central adiposity signal by binding to hypothalamic and other brain insulin receptors. Entry of circulating insulin into the CNS is accomplished by a saturable receptor-mediated transendothelial transport process (1). It has been shown that hyperinsulinemic, insulin-resistant, and hyperphagic obese Zucker rats exhibit a reduction in the number of brain capillary insulin receptors, which parallels the reduction in other peripheral tissues (5). This insulin receptor down-regulation resulted in a decrease in CSF insulin uptake. Lowering of brain insulin concentration is known to facilitate increased neuronal expression of neuropeptide Y (NPY) within the arcuate-paraventricular pathway involved in stimulation of food intake (6, 7, 8). This sequence of events likely plays a role in hyperphagic refeeding, especially since it has been demonstrated that NPY mRNA expression is markedly enhanced in obese compared with lean Zucker rats (9, 10). In our previous studies, we have demonstrated that attenuation of hyperinsulinemia by diazoxide (DZ), an inhibitor of insulin secretion, is associated with enhanced insulin sensitivity and decreased rate of weight gain in obese Zucker rats (11, 12). These data suggest that insulin resistance and obesity occur, at least in part, as a result of hyperinsulinemia. Since insulin acts centrally to reduce body weight (1), a plausible hypothesis is that the obesity state and hyperphagia observed in Zucker rats are in part due to insulin resistance in the brain, as manifested by reduced brain capillary insulin binding, which is thought to mediate the transport of insulin into the brain. Based on our previous results, we hypothesize that this reduction in brain capillary insulin binding is caused by hyperinsulinemia. Thus, we predict that attenuation of hyperinsulinemia by DZ in obese Zucker rats would partially reverse the impaired brain capillary insulin binding, leading to increased insulin uptake into the brain and reduced food intake and body weight. The present study tested these hypotheses.

	Materials and Methods

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Seven-week-old female obese Zucker (fa/fa) rats weighing 180–252 g and female Zucker lean (Fa/Fa) rats weighing 124–148 g were used in this study. Animals were obtained at age 6 weeks from Charles River Laboratories, Inc. (Wilmington, MA). They were phenotyped on the basis of body weight (BW) at 4 weeks of age. They were housed in individual stainless steel hanging metabolic cages and maintained on a 12-h light, 12-h dark cycle in a temperature-controlled room. The animals were fed ground rat chow (R.M.H. 3000, Agway, NY) and water ad libitum and were weighed daily. Obese and lean rats were divided into two subgroups of 24–28 animals per group: DZ-treated and vehicle (C, control) subgroups. DZ (150 mg/kg/day) was administered twice daily by gavage needle using Proglycem pediatric suspension 50 mg/ml (kindly provided by Baker-Norton Pharmaceuticals, Miami, FL). C groups were treated with an equivalent volume of vehicle suspension twice daily.

At the end of a 4-week treatment period, the DZ and C groups were each subdivided into fasted (FD, 18-h overnight fast) and free-feeding (FF) experimental subgroups. The following day (0900–1100), rats were anesthetized with ip injections of ketamine (65–100 mg/kg BW) and xylazine (10 mg/kg BW). CSF samples were obtained from the cisterna magna (cisternal puncture) while animals were secured in a Kopf stereotaxic apparatus as described previously (13). Plasma samples were obtained from a terminal cardiac puncture. Rats were euthanized by decapitation, and brain tissues were harvested for separation of cerebral cortices. The animal procedures were approved by the University of Tennessee Medical Center Animal Care Committee.

Assays
Plasma glucose and insulin and CSF insulin. Glucose was measured by the glucose oxidase method (Sigma Chemical Co., St Louis, MO), and plasma insulin (IRI) concentration was determined by RIA, using a double-antibody method (Linco Research, Inc., St Louis, MO). CSF samples containing more than 100 red cells/mm³ or having a volume less than 50 µl were not assayed. CSF insulin (IRI) was measured with rat double antibody method (Linco Research, Inc.) in an assay modified to produce higher sensitivity. This assay can detect 0.0036 pmol/ml with 95% confidence.

Plasma leptin. Leptin concentration was determined by RIA, using a double-antibody method (Linco Research, Inc.).

Binding assay
The isolation of brain capillaries
Brain capillaries were isolated using a modification of the method of Betz (14). After decapitation, brains were dissected on ice and the cerebral cortex was separated from the brain stem. Specimens were placed in ice-cold buffer A, consisting of Tissue Culture Medium 199, 1% antibiotic solution (penicillin, streptomycin, and Amphotericin), and 5% FCS, pH 7.4. The cerebral cortices were then homogenized in 5 volumes of buffer, using a hand-held homogenizer with a large clearance (0.25 mm; Kontes, Vineland, NJ). The homogenate suspension was centrifuged at 1,000 x g for 10 min at 4 C, the supernatant was discarded again, and after rinsing the pellet with buffer A, it was resuspended in a 2-fold volume excess of buffer A and passed over a 118-µm nylon mesh. The filtrate then was poured over a glass bead column (0.45- to 0.55-mm beads, B. Braun Biotech Intl., Allentown, PA) with the beads suspended over a 41-µm mesh, and the column was washed with 200 ml of buffer A. The suspension of glass beads and buffer was swirled with a glass rod for 2–3 min, after which the buffer containing the capillaries was aspirated from above the glass beads and transferred into a 30-ml centrifuge tube. The suspension was centrifuged at 1,000 x g for 5 min, resulting in a pellet of relatively pure brain capillaries. The pellet was washed twice with iced buffer B (118 mM NaCl, 4.7 mM KCI, 5.0 mM MgCl, 1 mM EGTA, 30 mM HEPES, 25 mg/liter bacitracin, 12.5 mg/liter N-ethylmaleimide, pH 7.9) after the removal of the supernatant. The capillaries were collected after each wash by centrifugation at 1,000 x g for 5 min at 4 C. The purity of the final preparation was verified by light microscopy (>90% pure brain capillaries). The pellet contents were resuspended in approximately 1.0 ml of buffer B, placed in 1.5-ml centrifuge tubes, and frozen at -70 C until assay.

Insulin binding to brain capillaries. One hundred microliters of brain capillary suspension were incubated for 29 h at 4 C with 0.1 pmol/ml Receptor grade Tyr^A14125I-Insulin (366 µCi/µg, New England Nuclear, Boston, MA) in the absence and presence of increasing concentrations of unlabeled insulin in a total volume of 1.0 ml. Triplicate aliquots of 200 µl of the suspensions at each concentration point were then centrifuged at 13,000 x g for 1 min. The pellet was then rinsed with iced buffer B with BSA and centrifuged again for 1 min at 13,000 x g. The microfuge tube tips containing the tissue pellet were cut off and counted on a -counter. Nonspecific binding was determined in the presence of 3.34 nmol/ml unlabeled insulin. Specific binding was defined as the difference between total bound in the presence of 0.1 pmol/ml [¹²⁵I]insulin and nonspecific binding, expressed as percent of total [¹²⁵I]insulin added. All data were corrected for nonspecific binding. The protein content of the tissue pellet was determined according to the method of Lowry et al. (15). Insulin binding was expressed as femtomoles/mg protein and by Scatchard analysis (16). Binding affinity and capacity were determined by a two-site Scatchard analysis using the NIH Ligand program (Ligand PC version 3.1) (17, 18).

To provide sufficient quantities of tissue for determination of specific binding, brain capillaries from two rats were pooled for each single determination of specifically bound insulin. All plasma and CSF values and BWs presented are derived from individual animals. Data were expressed as specifically bound insulin (femtomoles) per mg protein.

Chemicals
Crystalline porcine insulin and bovine albumin (fraction V) RIA grade were obtained from Sigma Chemical Co.. Mono ¹²⁵I-(Tyr^A14) insulin (366 µCi/ug) was obtained from New England Nuclear-Dupont (Boston, MA).

Statistical analysis
The reported values represent the mean ± SEM. Statistical analysis of subgroups was performed by one-way ANOVA, with significant differences between means determined by post hoc analysis using Dunnett’s multiple range test at P < 0.05.

	Results

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Table 1 shows weight and food intake in FD and FF obese and lean Zucker rats. Control obese rats had a higher initial weight and greater weight gain over the 4-week observation period than lean animals (Table 1). Final BW and average weight gain among DZ obese animals were decreased compared with control obese rats (P < 0.001), whereas DZ treatment had no effect on rate of weight gain in lean animals. When food intake was measured throughout the treatment period, control obese rats consumed larger amounts of food than control lean animals (P < 0.01). DZ obese rats demonstrated a marked reduction ( 40%) in food intake compared with control obese (P < 0.01) whereas DZ treatment had no significant effect on the amount of caloric intake in lean animals (Table 1).

Figures 1 and 2 show specific brain capillary insulin receptor binding and Scatchard plots of brain capillary insulin binding from obese and lean animals in FD and FF states, respectively. Obese rats showed significantly lower specific insulin receptor binding than lean rats, consistent with receptor down-regulation by hyperinsulinemia (Table 2). Insulin receptor binding in both DZ obese and DZ lean animals was higher than in their respective controls. When insulin binding data were analyzed using a two-site model, control lean and obese rats demonstrated similar affinity constants for both high- and low-affinity receptors (Table 3). Lean animals showed higher binding capacity of the high-affinity (R₁) and low-affinity (R₂) populations of receptors as compared with control obese. DZ obese and lean animals demonstrated greater high-affinity binding capacity (R₁) than their respective controls (P < 0.01). Similarly, binding capacity of low-affinity receptor sites (R₂) was significantly increased in DZ obese and lean animals (P < 0.01).

View larger version (18K): [in this window] [in a new window]   Figure 1. Specific insulin binding to brain capillaries in the obese [DZ and control (C)] and lean (DZ and C) Zucker rats. Data are expressed as femtomoles insulin bound/mg protein in FD (left) and FF (right) animals. Data are mean ± SEM. *, P < 0.05 (DZ lean vs. C lean); **, P < 0.01 (DZ obese vs. C obese); +, P < 0.01 (C obese vs. C lean).

View larger version (20K): [in this window] [in a new window]   Figure 2. Brain capillary insulin binding data are presented as Scatchard plots in obese [DZ and control (C)] and lean (DZ and C) Zucker rats. Insulin binding data are expressed as picomoles/mg protein in FD (top) and FF (bottom).

	Discussion

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Tenenbaum et al. (19) demonstrated that obese Zucker rats may have a propensity to hypersecrete insulin as early as 1 week of age, while hyperphagia of obese rats begins to emerge as early as 17 days of age (20). Obese rats continue to ingest more food than lean rats with the greatest trend to hyperphagia occurring between 5 and 15 weeks of age, overlapping the period of basal insulin surge. However, this hyperphagia does not appear to be necessary for the early increase in weight found in preweaning obese rats (21). In our previous studies, we found that DZ will reduce weight gain, improve insulin sensitivity, and reduce the rate of fat production, yet may not produce a major effect on feeding behavior in either obese or lean rats. In these studies, there may have been differences in feeding behavior between DZ obese and control obese groups earlier in the experiment when BW differences were emerging. However, food intake was measured during the final week of drug treatment when the shift in metabolic and behavioral controls may already have occurred. In the present study, food intake of DZ obese rats was markedly decreased as compared with control obese between 7 and 11 weeks of age.

While the increased peripheral insulin resistance in obese Zucker rats is indisputable, the effect of circulating insulin concentration on the CNS and feeding behavior is less well understood. This has been further complicated by the discovery of leptin, an obese gene product, that is believed to play a major role in regulation of NPY, a key orexigenic peptide in hypothalamus (22, 23). In our study, the plasma leptin levels were significantly higher in obese than lean rats, and DZ treatment resulted in significant suppression of leptin levels in obese and lean rats, which paralleled reduction in plasma insulin levels and BW consistent with observations in other studies (24, 25). Since obese Zucker rats have an underlying mutation in the leptin receptor (26, 27), they may provide a unique model to examine the role of insulin regulation of feeding independent of leptin physiological pathway.

Evidence for both saturable and specific uptake of insulin into the CNS suggests that receptor-mediated, transendothelial transport is important in the delivery of insulin into the brain (28). While CSF sampling may detect only insulin that has already passed through the brain tissue, the evaluation of relationship between steady-state levels of insulin in CSF and those in plasma (CSF/plasma ratio) provides an indirect measure of the rate of CSF insulin uptake from plasma. Israel et al. (29) evaluated the relationship between plasma insulin and CSF insulin uptake by raising the plasma insulin concentrations beyond the physiological range using continuous subcutaneous insulin infusion in Osborne-Mendel rats. They demonstrated that this relationship is curvilinear across a broad range of insulin levels, falling with increasing plasma levels, i.e. the fraction of the plasma insulin level present at steady state in CSF falls as plasma levels are raised beyond the physiological range due to a saturable transport process. This implies that CSF insulin uptake is more efficient at low than high plasma insulin levels. Similarly, CSF/plasma ratio is reduced to less than 50% of that of lean (Fa/Fa) controls, both in the basal state and during hyperinsulinemia induced by subcutaneous infusion of insulin (30), suggesting a reduction in the efficiency of insulin delivery across the blood-brain barrier in the fa/fa obese rats. Consistent with this hypothesis, brain capillary insulin binding is reduced in proportion to the decrease in CSF/plasma ratio in the obese rat (5). Ikeda et al. (4) demonstrated that infusion of a standard dose of insulin into the third ventricles of 4-month-old obese and lean Zucker rats resulted in a decrease in feeding in only the lean animals. They suggested that the lack of anorectic effect of insulin in obese animals was due to a defect in postreceptor mechanisms. In our study, attenuation of insulin secretion was associated with an up-regulation of brain capillary insulin receptor binding in obese and lean Zucker rats, accompanied by a significant increase in CSF insulin levels and CSF/plasma insulin ratio and a marked reduction in daily caloric intake in DZ-treated obese animals in both FD and FF states. The observed increase in CSF insulin levels despite a marked reduction in plasma insulin concentrations in obese animals may, in part, be due to enhanced brain capillary insulin binding and improved efficiency of transendothelial transport process. However, there was no significant increase in brain capillary insulin receptor affinity in DZ obese rats. Therefore, a direct effect of DZ on mechanism(s) regulating insulin clearance from CSF cannot be completely ruled out. On the other hand, DZ treatment did not significantly affect concentration of CSF insulin in lean animals despite an increased CSF/plasma insulin ratio and enhanced brain capillary insulin binding capacity. The latter finding may be due to the fact that the circulating insulin concentration of DZ lean animals was insufficient to induce a measurable change in CSF insulin concentration despite the enhanced insulin sensitivity. Consequently, the observed modest reduction in food intake in these animals was not statistically significant.

In examining the effect of DZ on feeding behavior in obese Zucker rats, Maggio and Vasselli (31) demonstrated a decreased food intake and reduced rate of weight gain in 9-week-old obese Zucker rats, suggesting that insulin hypersecretion plays a major role in producing hyperphagia in growing Zucker rats. Further, Rouru et al. (32) evaluated the effect of an antihyperglycemic agent, metformin (dimethyl-biguanide), on plasma insulin, food intake, and weight gain in hyperinsulinemic obese Zucker rats. They demonstrated that 2-week treatment of obese animals with metformin resulted in significant reduction in plasma insulin, food intake, and BW. However, the lean animals displayed only a transient decrease in food intake without a significant change in BW. These findings suggest that the anorectic effect of metformin may also be due to improved sensitivity of brain to circulating insulin.

In conclusion, this study suggests that attenuation of hyperinsulinemia in obese Zucker rats by DZ will enhance CNS insulin sensitivity and uptake, leading to a decrease in food intake and rate of weight gain. This implies that pharmacological modification of disturbed insulin metabolism by DZ may be therapeutically beneficial in hyperinsulinemic obesity. However, we cannot rule out a direct effect of DZ treatment on hypothalamic regulation of feeding behavior.

	Acknowledgments

 
We thank Ms. Charlotte Ketron for secretarial assistance in preparation of this manuscript.

	Footnotes

 
¹ This study was supported in part by a grant from Baker-Norton Pharmaceuticals, Inc., Miami, Florida.

Received September 11, 1998.

	References

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Alemzadeh, R. Articles by Holshouser, S. Search for Related Content PubMed PubMed Citation Articles by Alemzadeh, R. Articles by Holshouser, S.