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Endocrinology Vol. 141, No. 10 3630-3637
Copyright © 2000 by The Endocrine Society

ARTICLES

Diazoxide Restores ß3-Adrenergic Receptor Function in Diet-Induced Obesity and Diabetes

Richard S. Surwit1, Tonya M. Dixon1, Ann E. Petro, Kiefer W. Daniel and Sheila Collins

Departments of Psychiatry and Behavioral Sciences, and Pharmacology (T.M.D., S.C.), Duke University Medical Center, Durham, North Carolina 27710

Address all correspondence and requests for reprints to: Richard S. Surwit, Ph.D., Duke University Medical Center, Box 3842, Durham, North Carolina 27710.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We previously demonstrated that the expression and function of the adipocyte-specific ß3-adrenergic receptor (ß3AR) are significantly depressed in single gene and diet-induced rodent models of obesity. Furthermore, these models are relatively unresponsive to the antiobesity effects of ß3AR agonists. Because all of these models are hyperinsulinemic, we hypothesized that hyperinsulinemia could be responsible for this abnormality in ß3AR function. The goal of this study was to determine whether lowering insulin with the K-ATP channel agonist, diazoxide (Dz) would reverse the depressed expression and function of the ß3AR found in a model of diet-induced diabetes and obesity in C57BL/6J (B6) mice. B6 male mice were placed on either high fat (HF) or low fat experimental diets. After 4 weeks, HF-fed mice were assigned to a group: HF or HF containing disodium (R,R)-5-[2-([2-(3-chlorophenyl)-2-hydroxyethyl]-amino]propyl-1,3-benzodioxole-2,2-dicarboxylate (CL; 0.001%, wt/wt), Dz (0.32%, wt/wt), or their combination (CLDz). Dz animals exhibited significantly reduced plasma insulin levels as well as increased ß3AR expression and agonist-stimulated adenylyl cyclase activity in adipocytes. CLDz was more effective in reducing percent body fat, lowering nonesterified fatty acids, improving glucose tolerance, and reducing feed efficiency than either treatment alone.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IT HAS BEEN repeatedly demonstrated that various in-bred strains of mice are differentially susceptible to developing obesity on high fat (HF) diets (1, 2). One strain that is particularly vulnerable to this type of diet-induced obesity is the C57BL/6J (B6) mouse. When placed on a HF diet, B6 mice develop severe obesity, insulin resistance, and hyperglycemia (1, 3, 4). Furthermore, B6 mice develop diet-induced obesity without increased caloric consumption or a reduction in physical activity (5). Although the mechanism by which dietary fat induces diabetes and obesity in these mice is not understood, their obesity is characterized by adipocyte hyperplasia, particularly in the mesenteric fat pad and, at a molecular level, is accompanied by a loss of ß1- and ß3-adrenergic receptor (AR) expression and function in adipose tissue (6). Interestingly, this diet-induced impairment in the ßARs is quite similar to what we observed in monogenic models of obesity, such as Lepob, LepRdb, B6tub, and Cpefat (7, 8). In addition, we have shown that B6 mice, when raised on a HF diet, appear to be relatively refractory to the effects of a selective ß3AR agonist (6). As all three ßAR subtypes stimulate lipolysis (9, 10) and the induction of the uncoupling protein-1 (UCP1) gene in brown adipose tissue (BAT) (11) in response to catecholamines, we have hypothesized that defects in ß3AR, the most abundant AR in rodent adipose tissue, are responsible for the development of diet-induced obesity and diabetes in B6 mice.

The molecular basis for this diet-induced change in ßAR expression is not known. However, evidence is accumulating that hyperinsulinemia may play a role. Although genetic and dietary models of obesity display various endocrine abnormalities (12, 13), hyperinsulinemia is the one common feature among all of these models. In support of this idea, when differentiated 3T3-F442A mouse adipocytes were treated with insulin, ß3AR expression rapidly declined (14). In addition, a role for insulin in affecting ßAR function in adipocytes is supported by a series of studies showing that suppressing hyperinsulinemia with the K-ATP channel agonist, diazoxide (Dz), results in an improved ability to stimulate lipolysis and a significant loss of adipose tissue mass (15, 16).

The present study was designed to test the hypothesis that development of the obesity and diabetes syndrome in B6 mice raised on a HF diet is related to the hyperinsulinemia that arises in response to fat feeding. We hypothesized that suppressing the development of hyperinsulinemia in B6 mice with Dz would result in both an improvement in the diabetes/obesity phenotype and a reversal of the loss of ß3AR expression and function in adipocytes. A second objective of our study was to determine whether suppression of hyperinsulinemia would enable a selective ß3AR agonist to ameliorate this diet-induced syndrome.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and diets
Seventy-five 4-week-old B6 male mice were obtained from The Jackson Laboratory (Bar Harbor, ME). The Duke University animal care and use committee approved these animal studies. The animals were housed 5/cage in a temperature-controlled (22 C) room with a 12-h light, 12-h dark cycle (lights off at 1900 h). The HF and low fat (LF) experimental diets were manufactured by Research Diets (New Brunswick, NJ) and contain 58% and 11% of calories from fat, respectively. The compositions of these diets have been described in detail previously (4). A group of 60 mice were fed the HF diet for the first 4 weeks of the study; the remaining 15 mice were fed the LF diet. The mice assigned to the LF diet were maintained on this diet throughout the study as a reference group of lean control mice. At week 4, all HF-fed mice were assigned to 4 groups of 15 mice each. The first group remained on the HF diet throughout the study as the obese control group. The remaining 3 groups of mice were fed the HF diet containing the ß3AR agonist CL316,243 (CL), Dz, or a combination of the two compounds (CLDz). The CL concentration in the diets was 1 mg/kg (0.001%). This dose had originally been recommended by Dr. Thomas Claus, Lederle Laboratories (Pearl River, NY), and found to be differentially effective in reducing obesity in obesity-prone B6 and obesity-resistant A/J mice (17). The Dz concentration in the diets was 3.2 g/kg (0.32%), which was determined to be the maximally effective dose for weight reduction in dose-response pilot studies (data not shown).

Body weight, food intake, and feed efficiency
Animals were weighed weekly, and food consumption was measured per cage twice weekly until the diets were changed at week 4, whereupon body weight and food intake were determined daily excepting on weekends. The feed efficiency (grams of body weight gained per Cal consumed) was calculated on a per cage basis.

Glucose, insulin, and leptin
Samples for analysis of insulin, glucose, and leptin were collected as we have previously reported (4, 6) on day 24 (4 days before the diets were changed), on day 32 (4 days after the change), and biweekly thereafter. In all cases food was removed 8 h before samples were collected. Glucose was analyzed by the glucose oxidase method (Glucose Analyzer II, Beckman Coulter, Inc., Palo Alto, CA). Insulin and leptin concentrations were determined by double antibody RIA (Linco Research, Inc., St. Louis, MO). The insulin assay was based on a rat standard, and the leptin assay used a mouse standard.

Triglycerides and nonesterified fatty acids
At the termination of the study, a postprandial plasma sample was collected and analyzed for triglyceride and nonesterified fatty acid concentrations using kits from WAKO Diagnostics (Richmond, VA).

Tissue collection
After 4 weeks of drug treatment, a subset of 10 animals from each group was killed. The epididymal white adipose tissue (EWAT), retroperitoneal (RP) fat, interscapular brown adipose tissue (IBAT) fat pads, and gastrocnemius muscle were removed, trimmed, and weighed, and all tissues were flash-frozen in liquid nitrogen and stored at -80 C for later determination of ß3AR (EWAT), UCP1 expression (IBAT and RP), UCP2 and -3 expression (IBAT), as well as ß3AR-stimulated adenylyl cyclase activity (EWAT).

Percent body fat
The percent body fat was estimated from the weight of the epididymal fat pad. It has been shown that as a proportion of total body weight, epididymal fat pad weight is highly correlated with percent body fat (18, 19).

Glucose tolerance test
A subset of five animals from each group was injected ip with 0.5 g/kg glucose. At 30 min postinjection, a plasma sample was collected and analyzed for glucose content by the glucose oxidase method.

Glucose transport
Basal glucose transport into white adipose tissue and muscle was assessed following the method of Skillman and Fletcher (20) with minor modifications. Five mice from each group were injected ip with 50 mg/kg 2-deoxyglucose (20 µCi/kg [14C]2-deoxyglucose) mixed with 2-deoxyglucose (final SA, 66 µCi/mmol). Twenty minutes later the mice were killed by cervical dislocation. The thoracic cavity was opened, and 5 ml saline were perfused through the left ventricle with the right atrium cut to diminish the vascular 14C levels. The right epididymal and RP fat pads and gastrocnemius muscle were excised, rinsed, blotted, and weighed. The tissues were solubilized in 1 M NaOH at 37 C overnight. The samples were neutralized with 1 M HCl, and the 14C content was determined.

Isolation and analysis of RNA
Total cellular RNA was prepared using the tissues of six mice per group by the cesium chloride gradient method as detailed previously (21). For Northern blot hybridization, RNA was denatured by the glyoxal procedure, fractionated through 1.2% agarose gels, and blotted onto Biotrans (ICN Biomedicals, Inc., Costa Mesa, CA) nylon membranes (22). Radiolabeled probes were prepared by random primer synthesis (PrimeIT, Stratagene, La Jolla, CA) of the purified DNA fragments in the presence of [{alpha}-32P]deoxy-CTP to a specific activity of more than 2 x 109 dpm/µg DNA. A fragment specific for the mouse ß3AR was prepared as previously described (7). For mitochondrial UCP1, a 300-bp BglI fragment, provided by Dr. Leslie P. Kozak, was used (23). A rat complementary DNA probe for cyclophilin was used as an internal hybridization/quantitation standard. Blots were hybridized and washed as previously described (7, 24). The intensity of hybridization signals was quantified by PhosphorImager (ImageQuant/Storm, Molecular Dynamics, Inc., Sunnyvale, CA) and normalized to the values for cyclophilin.

Preparation of plasma membranes and adenylyl cyclase assay
Adipose tissue was isolated from each group of animals. The tissues from four mice per group were pooled and minced, then plasma membranes were prepared as previously described (7). Adenylyl cyclase activity was measured using established methods, and the cAMP formed was measured by RIA (25) using a polyclonal antiserum to cAMP (26). Protein concentrations were determined by the Bradford method (27).

Statistical analyses
Unless otherwise noted, data were analyzed using ANOVA. P < 0.05 was considered significant. Post-hoc comparisons were made using the least significant difference test. Analyses within groups at different time points were performed using t test for dependent samples. Data from LF animals were not included in statistical analyses.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Body weight, food intake, and feed efficiency
The body weights of all groups consuming the HF diet were similar at the end of the pretreatment period. After administration of the compounds for 4 weeks, ANOVA showed a significant effect of treatment on body weight (P < 0.001). As shown in Fig. 1Go, all three therapies reduced body weight gain compared with that of the HF control group. At the end of the study, the combination therapy was more effective than CL alone, but was not more effective than Dz alone. Immediately after the change from HF to HF plus drugs, there was a modest and brief (<4 days) decrease in food intake. However, ANOVA showed that total food intake tended to be influenced by treatment (P = 0.08). Animals fed the diet containing CLDz ate more. Figure 2Go illustrates that all treatments reduced feed efficiency compared with that of HF (ANOVA P < 0.001). Feed efficiency is the ratio of weight gained to calories consumed. It therefore reflects metabolic efficiency and is not confounded by caloric intake. CLDz was more effective than either CL or Dz in decreasing feed efficiency. Thus, even though the CLDz-treated animals showed similar decreases in fasting insulin, they gained less weight.



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  		Figure 1. The effect of LF ({diamondsuit}), HF ({blacksquare}), and HF diets containing CL ({square}), Dz ({circ}), or CLDz (•) on body weight of B6 mice. Mice were fed the HF diet for 4 weeks before starting the treatments. Treatments with different superscripts are significantly different: a vs. b, P < 0.001; a vs. c, P < 0.001; b vs. c, P < 0.01. Mice fed the LF diet served as a reference group of lean controls.

 


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  		Figure 2. The feed efficiency (body weight gain per Cal consumed) of B6 mice after consuming LF, HF, HF plus 0.001% CL, HF plus 0.32% Dz, or HF plus CLDz for 1 month. Treatments with different superscripts are significantly different: a vs. b, P < 0.001; a vs. c, P < 0.001; b vs. c, P < 0.05. Mice fed the LF diet served as a reference group of lean controls.

 
Estimate of percent body fat
The effect of treatment on percent body fat is shown in Fig. 3Go. The weight of the EWAT fat pad as a proportion of total body weight was used to estimate the percent body fat. Percent body fat was significantly affected by treatment (by ANOVA, P < 0.001). Importantly, CLDz was significantly more effective in decreasing percent body fat than either CL or Dz.



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  		Figure 3. An estimate of the percent body fat in B6 mice fed LF, HF, HF plus 0.001% CL, HF plus 0.32% Dz, or HF plus CLDz for 1 month. Percent body fat was estimated by the weight of epididymal fat as a proportion of the total body weight. See Refs. 17 and 18 for validation of estimate. Treatments with different superscripts are significantly different: a vs. b, P < 0.001; a vs. c, P < 0.001; b vs. c, P < 0.05. Mice fed the LF diet served as a reference group of lean controls.

 
Fat pad weight
The wet weights of RP and EWAT fat pads are shown in Table 1Go. Both fat pads were significantly affected by treatment (ANOVA P < 0.001 for both fat pads). RP fat pad weight was reduced by all treatments when compared with the HF group (P < 0.001 for all treatments). CLDz was found to be more effective in decreasing RP weight than CL (P < 0.01), but was as effective as Dz (P = .51). Similarly, all treatments reduced the wet weight of EWAT fat pads when compared with the HF group (P < 0.001 for all treatments). In addition, CLDz was significantly more effective than CL (P < 0.01) and tended to be more effective (P = 0.08) than Dz in reducing EWAT weight.


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  		Table 1. Leptin, plasma lipids, and fat pad weights

 
Insulin and glucose
The effects of these various treatments on fasting plasma insulin and glucose over the course of the study are illustrated in Fig. 4Go. Within 4 days there was a significant effect of treatment on plasma insulin, as shown by ANOVA (P < 0.001). Insulin levels (Fig. 4AGo) were significantly decreased in all treated groups compared with those in the HF group and compared with their respective pretreatment values. At the conclusion of the study, ANOVA indicated a significant effect of treatment on plasma insulin (P < 0.001). Insulin levels in the HF group continued to increase throughout the study, and they remained significantly higher than those with any of the treatments (P < 0.01 for all treatments). Initially, the insulin levels within the CL group decreased; however, by the end of the study they were similar to the pretreatment value, even though they were lower than those in the HF group. By contrast, insulin levels in the Dz and CLDz groups remained significantly below pretreatment levels throughout the study. There appeared to be no added benefit of CLDz.



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  		Figure 4. The effect of treatment on fasting plasma insulin (A) and glucose (B). Mice were fed a HF diet for 1 month before the treatment began. See Materials and Methods for details. Blood was collected before the treatment period started and biweekly thereafter. {diamondsuit}, LF; {blacksquare}, HF; {square}, HF plus 0.001% CL; {circ}, HF plus 0.32% Dz; •, HF plus CLDz. Treatments with different superscripts are significantly different on day 31: A) insulin: a vs. b, P < 0.001; a vs. c, P < 0.001; b vs. c, P < 0.05; B) glucose: a vs. b, P < 0.001; a vs. c, P < 0.001; b vs. c, P < 0.001. LF-fed mice served as a reference group of lean controls.

 
Within 4 days of instituting the drug regimens, there was a significant effect of treatment on plasma glucose (by ANOVA, P < 0.001). Figure 4BGo shows that glucose levels in all treatment groups dropped significantly compared with those in the HF control group. In addition, there was an immediate added benefit of CLDz of lowering plasma glucose levels. On day 4, glucose values in the CLDz group were significantly less than values with either treatment alone. The normalization in fasting plasma glucose values in the CL group was transient. Similar to the insulin levels in this group, plasma glucose had returned to the pretreatment values at the termination of the study. In contrast, fasting plasma glucose concentrations in the Dz and CLDz groups remained lower than pretreatment levels throughout the study.

Plasma lipids and leptin
Values for these parameters at the conclusion of the study are shown in Table 1Go. Treatment significantly affected plasma leptin (by ANOVA, P < 0.001). Leptin levels were lower in all groups compared with the HF control values. Although CL decreased leptin levels compared with the HF control values, it was less effective for lowering leptin levels than either Dz or CLDz (P < 0.001 for both comparisons).

Plasma triglycerides were also affected by treatment (P < 0.001). Although CL treatment significantly raised triglyceride levels compared with those in the HF control group (P < 0.05), Dz and CLDz lowered triglyceride levels (Dz, P < 0.01; CLDz, P < 0.001). In addition, all treatments were effective in lowering nonesterified fatty acids in plasma compared with levels in HF-fed mice (CL, P < 0.001; Dz, P < 0.05; CLDz, P < 0.001), but CLDz was significantly more effective than either CL (P = 0.01) or Dz (P < 0.001).

Glucose tolerance
The mice were challenged with an ip bolus of glucose. Figure 5Go shows the effect of treatment on glucose tolerance. CLDz was more effective than either CL or Dz in improving glucose tolerance. In animals treated with CLDz, plasma glucose returned to the postabsorptive level within 30 min after the challenge (98 mg/dl postabsorptive vs. 106 mg/dl postchallenge).



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  		Figure 5. The effect of treatment on glucose tolerance. Mice were injected with 0.5 g/kg glucose. Blood samples were collected 30 min later and analyzed for glucose. Treatments with different superscripts are significantly different: a vs. b, c, or d, P < 0.001; b vs. c, P < 0.01; b vs. d, P < 0.001; c vs. d, P < 0.05. LF-fed mice served as a reference group of lean controls.

 
Glucose transport into adipose and muscle tissues
The efficacy of treatment on glucose transport into EWAT and RP fat pads is shown in Fig. 6Go. There was a significant effect of treatment on glucose transport into EWAT (by ANOVA, P < 0.05). CL and CLDz improved glucose transport into EWAT compared with that in the HF control group, but treatment with Dz did not reach a statistically significant level. With regard to the effects of these compounds on glucose transport into RP, ANOVA revealed a trend (P = 0.07) toward a treatment effect. As shown in the figure, CL and CLDz enhanced glucose transport compared with that in the HF control group. However, there was no apparent effect of treatment on glucose transport into muscle tissues (data not shown).



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  		Figure 6. The effect of treatment on glucose transport into EWAT and RP adipose tissue as measured by the accumulation of [14C]2-deoxyglucose in the tissues. Treatments with different superscripts are significantly different: a vs. b, P < 0.05. The ANOVA for retroperitoneal fat was not statistically significant (P = 0.07). LF-fed mice served as a reference group of lean controls.

 
Adipocyte ß3AR function
We have previously shown that ß3AR-stimulated adenylyl cyclase activity in EWAT is severely blunted in B6 mice fed a HF diet (6). However, as the wet weight of the epididymal fat pads (and RP, not shown) from CL-, Dz-, and CLDz-treated animals were significantly less than those from their HF counterparts (Fig. 3Go), it suggested to us that lipolysis of previously stored lipids had occurred in each case. Therefore, we examined plasma membranes from EWAT for adenylyl cyclase activity in response to CL, as ßAR-stimulated cAMP production is the major proximal step regulating lipolysis (28). The means of three experiments in each condition are shown in Fig. 7Go. Nonlinear regression analysis revealed that all three curves were significantly different from each other (P < 0.0003). As previously observed in obese animals (6, 7), basal and ß3AR-stimulated adenylyl cyclase activities were significantly depressed in HF-fed B6 mice compared with those in LF animals (P < 0.0001). Treatment with Dz not only reduced plasma levels of insulin and glucose, but also restored the efficacy of the ß3AR to stimulate adenylyl cyclase (P < 0.0001). This is consistent with an increased ability to recruit stored lipids through this pathway. When we made similar measurements in adipocyte plasma membranes from CL- and CLDz-treated animals, we encountered a general decrease in adenylyl cyclase activity, which we have seen before in animals chronically treated with CL (6) (our unpublished data). The molecular basis of this phenomenon is still unclear, but is presumed to be some form of partial down-regulation of the signaling cascade.



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  		Figure 7. The stimulation of adenylyl cyclase activity by the ß3AR-selective agonist CL in membranes from animals fed the LF ({diamondsuit}), HF ({blacksquare}), or Dz ({circ}) diets. The assays were incubated for 10 min. The cAMP produced was measured by RIA as described in Materials and Methods. The data are expressed as picomoles of cAMP produced per mg membrane protein/min incubation. Curves represent the mean of three experiments for each condition. Nonlinear regression analysis revealed that all three curves were significantly different from each other (P < 0.0003).

 
ß3AR expression in EWAT
We next determined whether the observed changes in ß3AR function in adipose tissue, as defined by adenylyl cyclase activity, were accompanied by concomitant alterations in ß3AR messenger RNA (mRNA) expression. The HF diet significantly reduced the expression of ß3AR mRNA in EWAT compared with that in the LF control group (P < 0.01), as observed previously (6). Figure 8Go shows that animals treated with Dz alone expressed ß3AR mRNA at levels comparable to the LF-fed controls. Similar to our previous studies (6), B6 mice treated with CL failed to restore ß3AR expression or function (not shown). Figure 8Go also shows that there was no improvement in ß3AR mRNA levels in CLDz-treated animals. This result is confounding in view of the fact that WAT from these animals exhibited the greatest response to treatment in terms of decreased adipose tissue mass. However, despite the fact that we do not yet understand the apparent loss of mRNA expression of the ß3AR that is routinely observed in ß3AR agonist-treated animals, the physiological effect on total body weight and fat mass is clearly improved in these combination-treated animals.



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  		Figure 8. The effect of treatment on ß3AR mRNA levels in EWAT. Forty micrograms of total cellular RNA from EWAT were fractionated through 1.2% agarose gels and blotted as described in Materials and Methods. The blot was probed with {alpha}-32P-labeled ß3AR and cyclophilin. Amounts of ß3AR mRNA were determined with a Molecular Dynamics, PhosphorImager and were normalized to cyclophilin mRNA levels. Treatments with different superscripts are significantly different: a vs. b, P < 0.05. LF-fed mice served as a reference group of lean controls.

 
Regulation of brown adipocyte UCP1
The expression of UCP1 is specific to brown adipocytes and thus serves as a marker for the presence and thermogenic activity of IBAT (29). We (6) and others (30, 31) previously showed that the ability of ß3AR agonists to prevent or reverse obesity is linked to the expansion of brown adipocytes and UCP1 expression, particularly in WAT depots, and varies between strains of mice (6). Therefore, we examined how the expression of UCP1 was affected in various adipose depots in B6 mice in response to a HF diet and subsequent treatment with Dz, CL, and CLDz. As shown in Fig. 9Go and consistent with previous studies (6, 32), levels of UCP1 mRNA were increased in IBAT in response to the HF diet compared with those in the LF control group (P < 0.01). Animals treated with either CL or Dz exhibited a greater increase in UCP1 expression (P < 0.05 and P < 0.01, respectively), whereas animals treated with the combination therapy showed a markedly greater elevation of UCP1 mRNA levels (P < 0.001) compared with the HF control. In the RP depot, which consists of a mixture of white and brown adipocytes (33), there was a modest decrease in UCP1 expression in response to HF feeding compared with the LF control (P > 0.05), consistent with our previous findings (6). In HF animals receiving CL, Dz, or CLDz, only those treated with the combination therapy showed a significant increase in UCP1 expression (P < 0.05). Only HF-fed B6 mice treated with Dz showed a significant increase in UCP3 expression in IBAT (P < 0.05; data not shown), whereas there was no effect of treatment on UCP2 expression in IBAT (data not shown). It should be noted that increases in mRNA usually, but do not necessarily, indicate corresponding changes in protein levels. Unfortunately, we were unable to measure protein levels of ß3AR or UCP1.



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  		Figure 9. Effect of treatment on UCP1 levels in IBAT and RP. Methods were previously described (8 ), except that blots were probed with {alpha}-32P-labeled UCP1 and cyclophilin. Treatments with different superscripts are significantly different: IBAT: a vs. b, P < 0.05; a vs. c, P < 0.001; b vs. c, P < 0.01; RP: a vs. b, P < 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The K-ATP channel agonist, Dz, has been shown to greatly attenuate obesity in the Zucker fatty rat and facilitate weight loss in obese humans (15, 16). Because we have previously shown that B6 mice as well as other hyperinsulinemic models of obesity exhibit decreased ß3AR expression and function (6, 8), and in vitro studies reported that insulin could adversely affect the expression of ß3ARs (14), we postulated that the effects of Dz on adipose tissue mass and glucose metabolism could be mediated by decreased circulating insulin in Dz-treated animals. Furthermore, the B6 mice raised on a HF diet are comparatively insensitive to treatment with the selective ß3AR agonist, CL (6). In the present study, treatment with Dz resulted in an increase in the expression of ß3AR and an improvement in the function, as evidenced by increased cAMP production in response to selective ß3AR agonist stimulation. In addition, animals treated with Dz showed an increase in expression of UCP1 in both IBAT and RP fat pads. Although this increase in UCP1 expression might be a direct effect of increased ßAR function, the vasodilatory effects of Dz may have resulted in decreased body temperature and provoked a compensatory thermogenic response in BAT. In addition to effects on ßAR function, recent data suggest that Dz has direct effects on Ca+2 influx through the adipocyte sulfonyl urea receptor 1 and the K-ATP channel, resulting in increased lipolysis (34). However, the size of these direct effects was relatively small, making it unlikely that they account for all of the Dz-induced changes in diet-induced obesity and diabetes described here.

Our hypothesis that the antiobesity effects of Dz are due to increased ß3AR expression and function would predict that the effects of CL combined with Dz would be synergistic. Indeed, we observed that CLDz was more effective in reducing feed efficiency, decreasing percent body fat, reducing circulating FFAs, and improving glucose tolerance than either Dz or CL alone. However, with the exception of feed efficiency, the combined effects appeared to be additive, rather than synergistic. It is quite likely that the failure to observe a synergistic effect was due to the doses of CL and Dz chosen for the study, both of which were maximally effective when used alone (6) (our unpublished pilot studies). A lower dose of one or both might have produced a synergistic reaction. Further studies will be needed to examine this issue. Nevertheless, CLDz improved glucose tolerance and lowered feed efficiency as well as weight of EWAT and RP fat pads without further lowering insulin, suggesting that these effects were independent of the direct effect of Dz on plasma insulin per se.

The effect of CLDz on glucose metabolism was accompanied by a significant decrease in feed efficiency and an increase in glucose transport in EWAT and RP, but not in muscle. Increased glucose transport in EWAT and RP was accompanied by a decrease in the size of these fat pads as well. This suggests that changes in insulin sensitivity and metabolic activity in fat rather than muscle are critical to the development of diabetes in the B6 mouse model. This is in agreement with previous work in which Dz was shown to increase glucose transport in fat (15) and in studies in which CL has been shown to increase glucose transport (35, 36). Further support for a significant role of adipose tissue in glucose disposal comes from studies in transgenic mice that overexpress GLUT4 selectively in adipose tissue, which exhibit a marked attenuation in streptozotocin-induced dia-betes (37).

Our findings thus add to the growing literature that emphasizes the importance of the adipocyte in insulin resistance and suggest a primary role for hyperinsulinemia in insulin-resistant syndromes. Specifically, we propose that in the B6 mouse, HF diets promote hyperinsulinemia, and this, in turn, leads to insulin resistance, in part through diminished ßAR function in adipose tissue. Although the increased circulating leptin associated with increased adipose tissue should lead to increased sympathetic outflow and thermogenesis (38), insulin-induced down-regulation of ßAR function in adipose tissue disrupts the normal neuroendocrine feedback that regulates adipocyte function (6). Thus, we have speculated that hyperleptinemia in obesity results at least in part from peripheral ßAR dysfunction (6). Hyperleptinemia, in turn, could lead to down-regulation of the central neuropeptide receptors involved in thermogenesis and appetite control. Reversal of hyperinsulinemia with diazoxide, then, may improve not only ßAR function, but central neuropeptide function as well. Although the mechanism by which a HF diet initiates hyperinsulinemia is not known, we have previously shown that HF feeding attenuates the insulin response to glucose in isolated islets from B6 mice, whereas the insulin response to lipid is less affected (39). Further research is needed to determine whether this defect in islet function is correctable with Dz, and whether the effect of Dz to improve adipocyte ßAR function in our model is due primarily to reductions in insulin levels. Nevertheless, the data presented here suggest that combining an insulin-suppressing agent such as Dz with a ß3AR agonist could prove to be a more potent therapy for diabetes and obesity than either treatment alone.


   Acknowledgments
 
We thank Dr. Tom Gettys for the gift of the cAMP antisera. We also thank Paul Blackwelder and Jason Hampton for their technical assistance.


   Footnotes
 
1 These authors contributed equally to this manuscript. Back

Received March 28, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Surwit RS, Kuhn CM, Cochrane C, McCubbin JA, Feinglos MN 1988 Diet-induced type II diabetes in C57BL/6J mice. Diabetes 9:1163–1167
  2. West DB, Boozer CN, Moody DL, Atkinson RL 1992 Dietary obesity in nine inbred mouse strains. Am J Physiol 262:R1025–R1032
  3. Surwit RS, Seldin MF, Kuhn CM, Cochrane C, Feinglos MN 1991 Control of expression of insulin resistance and hyperglycemia by different genetic factors in diabetic C57BL/6J. Diabetes 40:82–87[Abstract]
  4. Surwit RS, Feinglos MN, Rodin J, Sutherland A, Petro AE, Opara EC, Kuhn CM, Rebuffe-Scrive M 1995 Differential effects of fat and sucrose on the development of obesity and diabetes in C57BL/6J and A/J mice. Metabolism 44:645–651[CrossRef][Medline]
  5. Brownlow BS, Petro A, Feinglos MN, Surwit RS 1996 The Role of Motor Activity in Diet-Induced Obesity in C57BL/6J Mice. Physiol Behav 60:37–41[CrossRef][Medline]
  6. Collins S, Daniel KW, Petro AE, Surwit RS 1997 Strain-specific response to ß3-adrenergic receptor agonist treatment of diet-induced obesity in mice. Endocrinology 138:405–413[Abstract/Free Full Text]
  7. Collins S, Daniel KW, Rohlfs EM, Ramkumar V, Taylor IL, Gettys TW 1994 Impaired expression and functional activity of the ß3- and ß1-adrenergic receptors in adipose tissue of congenitally obese (C57BL/6J ob/ob) mice. Mol Endocrinol 8:518–527[Abstract]
  8. Collins S, Daniel KW, Rohlfs EM 1999 Depressed expression of adipocyte ß-adrenergic receptors is a common feature of congenital and dietary obesity in rodents. Intl J Obes 23:669–677
  9. Galitzky J, Reverte M, Portillo M, Carpene C, Lafontan M, Berlan M 1993 Coexistence of ß1-, ß2-, and ß3-adrenoceptors in dog fat cells and their differential activation by catecholamines. Am J Physiol 264:E403–E412
  10. Galitzky J, Carpéné C, Bousquet-Mélou A, Berlan M, Lafontan M 1995 Differential activation of ß1-, ß2- and ß3- adrenoceptors by catecholamines in white and brown adipocytes. Fundam Clin Pharmacol 9:324–331[Medline]
  11. Rohlfs EM, Daniel KW, Premont RT, Kozak LP, Collins S 1995 Regulation of the uncoupling protein gene (Ucp) by ß1, ß2, ß3-adrenergic receptor subtypes in immortalized brown adipose cell lines. J Biol Chem 270:10723–10732[Abstract/Free Full Text]
  12. Coleman DL 1982 Diabetes-obesity syndromes in mice. Diabetes 31:1–6[Abstract]
  13. Coleman DL, Eicher EM 1990 Fat (fat) and tubby (tub): two autosomal recessive mutation causing obesity syndromes in the mouse. J Hered 81:424–427[Abstract/Free Full Text]
  14. Fève B, Elhadri K, Quignard-Boulange A, Pairault J 1994 Transcriptional down-regulation by insulin of the ß3-adrenergic receptor expression in 3T3–F442A adipocytes: a mechanism for repressing the cAMP signaling pathway. Proc Natl Acad Sci USA 91:5677–5681[Abstract/Free Full Text]
  15. Alemzadeh R, Jacobs W, Pitukcheewanont P 1996 Antiobesity effect of diazoxide in obese Zucker rats. Metabolism 45:334–341[CrossRef][Medline]
  16. Alemzadeh R, Langley G, Upchurch L, Smith P, Slonim AE 1998 Beneficial effect of diazoxide in obese hyperinsulinemic adults. J Clin Endocrinol Metab 83:1911–1915[Abstract/Free Full Text]
  17. Collins S, Daniel KW, Petro AE, Surwit RS, Andersen PH 1997 Combination treatment of dietary obesity in mice with growth hormone and a ß3AR agonist promotes greater weight loss and increased UCP1 expression than either agent alone. Obes Res 5:6S
  18. Rogers P, Webb GP 1980 Estimation of body fat in normal and obese mice. Br J Nutr. 43:83–86
  19. Eisen EJ, Leatherwood JM 1981 Predicting percent fat in mice. Growth 45:100–7[Medline]
  20. Skillman CA, Fletcher HP 1986 2-Deoxyglucose tissue levels and insulin levels following tolazamide dosing in normal and obese mice. Exp Clin Endocrinol 87:288–298[Medline]
  21. Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ 1979 Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294–5299[CrossRef][Medline]
  22. Collins S, Caron MG, Lefkowitz RJ 1988 ß2-Adrenergic receptors in hamster smooth muscle cells are transcriptionally regulated by glucocorticoids. J Biol Chem 263:9067–9070[Abstract/Free Full Text]
  23. Kozak L, Britton J, Kozak U, Wells J 1988 The mitochondrial uncoupling protein gene. J Biol Chem 263:12274–12277[Abstract/Free Full Text]
  24. Thomas PS 1980 Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci USA 77:5201–5205[Abstract/Free Full Text]
  25. Harper JF, Brooker G 1975 Femtomole sensitive radioimmunoassay for cyclic AMP and cyclic GMP after 2'0-acetylation by acetic anhydride in aqueous solution. J Cyclic Nucleotide Res 1:207–218[Medline]
  26. Gettys TW, Ramkumar V, Uhing RJ, Seger L, Taylor IL 1991 Alterations in mRNA levels, expression, and function of GTP-binding regulatory proteins in adipocytes from obese mice (C57BI/6J-ob/ob). J Biol Chem 266:15949–15955[Abstract/Free Full Text]
  27. Bradford M 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72:248–254[CrossRef][Medline]
  28. Robinson G, Butcher R, Sutherland E 1971 Cyclic AMP. Academic Press, New York
  29. Bouillaud F, Ricquier D, Mory G, Thibault J 1984 Increased level of mRNA for the uncoupling protein in brown adipose tissue of rats during thermogenesis induced by cold exposure or norepinephrine infusion. J Biol Chem 259:11583–11586[Abstract/Free Full Text]
  30. Champigny O, Ricquier D, Blondel O, Mayers RM, Briscoe MG, Holloway BR 1991 ß3-Adrenergic receptor stimulation restores message and expression of brown-fat mitochondrial uncoupling protein in adult dogs. Proc Natl Acad Sci USA 88:10774–10777[Abstract/Free Full Text]
  31. Himms-Hagen J, Phil D, Danforth Jr E 1996 The potential role of ß3-adrenoceptor agonists in the treatment of obesity and diabetes. Curr Opin Endocrinol Diabetes 3:59–65
  32. Surwit RS, Wang S, Petro AE, Sanchio D, Raimbault S, Ricquier D, Collins S 1998 Diet-induced changes in uncoupling proteins in obesity-prone and obesity-resistant strains of mice. Proc Natl Acad Sci USA 95:4061–4065[Abstract/Free Full Text]
  33. Cousin B, Cinti S, Morroni M, Raimbault S, Ricquier D, Penicaud L, Casteilla L 1992 Occurrence of brown adipocytes in rat white adipose tissue: molecular and morphological characterization. J Cell Sci 103:931–942[Abstract/Free Full Text]
  34. Shi H, Moustaid-Moussa N, Wilkison WO, Zemel MB 1999 Role of the sulfonylurea receptor in regulating human adipocyte metabolism. FASEB J 13:1833–1838[Abstract/Free Full Text]
  35. Liu X, Perusse F, Bukowiecki L 1998 Mechanisms of the antidiabetic effects of the ß3-adrenergic agonist CL-316,243 in obese Zucker-ZDF rats. Am J Physiol 43:R1212–R1219
  36. de Sousa C, Hirshman M, Horton E 1997 CL-316,243, a ß3-specific adrenoceptor agonist, enhances insulin-stimulated glucose disposal in nonobese rats. Diabetes 46:1257–1263[Abstract]
  37. Tozzo E, Gnudi L, Kahn BB 1997 Amelioration of insulin resistance in streptozocin diabetic mice by transgenic overexpression of Glut 4 driven by an adipose-specific promoter. Endocrinology 138:1604–1611[Abstract/Free Full Text]
  38. Collins S, Kuhn CM, Petro AE, Chrunyk B, Swick AG, Surwit RS 1996 Leptin and fat regulation. Nature 380:677[CrossRef][Medline]
  39. Lee S, Opara E, Surwit R, Feinglos M, Akwari O 1995 Defective glucose-stimulated insulin release from perifused islets of C57BL/6J mice. Pancreas 11:206–211[Medline]



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