AJP - Endo AJP: Cell Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Endocrinol Metab 290: E1227-E1236, 2006; doi:10.1152/ajpendo.00344.2005
0193-1849/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via ISI Web of Science (4)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kim, H.
Right arrow Articles by LeRoith, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kim, H.
Right arrow Articles by LeRoith, D.

Effect of adipocyte beta3-adrenergic receptor activation on the type 2 diabetic MKR mice

Hyunsook Kim,1,3,* Patricia A. Pennisi,1,* Oksana Gavrilova,2 Stephanie Pack,2 William Jou,2 Jennifer Setser-Portas,1 Joyce East-Palmer,1 Yan Tang,4 Vincent C. Manganiello,4 and Derek LeRoith1

1Diabetes Branch and 2Mouse Metabolism Core Laboratory, National Institute of Diabetes, Digestive and Kidney Diseases; 3Beltsville Human Nutrition Center, Agricultural Research Service-United States Department of Agriculture, Beltsville; and 4Pulmonary Critical Care Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland

Submitted 29 July 2005 ; accepted in final form 2 January 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The antiobesity and antidiabetic effects of the beta3-adrenergic agonists were investigated on nonobese type 2 diabetic MKR mice after injection with a beta3-adrenergic agonist, CL-316243. An intact response to acute CL-316243 treatment was observed in MKR mice. Chronic intraperitoneal CL-316243 treatment of MKR mice reduced blood glucose and serum insulin levels. Hyperinsulinemic euglycemic clamps exhibited improvement of the whole body insulin sensitivity and glucose homeostasis concurrently with enhanced insulin action in liver and adipose tissue. Treating MKR mice with CL-316243 significantly lowered serum and hepatic lipid levels, in part due to increased whole body triglyceride clearance and fatty acid oxidation in adipocytes. A significant reduction in total body fat content and epididymal fat weight was observed along with enhanced metabolic rate in both wild-type and MKR mice after treatment. These data demonstrate that beta3-adrenergic activation improves the diabetic state of nonobese diabetic MKR mice by potentiation of free fatty acid oxidation by adipose tissue, suggesting a potential therapeutic role for beta3-adrenergic agonists in nonobese diabetic subjects.

insulin-like growth factor I receptor mutation; type 2 diabetes

beta3-ADRENERGIC RECEPTORS (beta3-AR) are primarily expressed in brown (BAT) and white adipose tissue (WAT) (3, 4, 11, 27). Their activation with selective agonists stimulates lipolysis and release of fatty acids in WAT and also activation of thermogenesis in BAT (1, 19, 28, 34). Reduction of murine beta3-AR number in adipose tissue of obese mice and Trp64Arg missense mutation of beta3-AR in obese humans both indicate a possible role of beta3-AR in obesity-related insulin resistance (12, 21, 38). However, the actual physiological role of beta3-AR in humans is not clear due to the lack of a highly selective beta3-AR agonist despite continuous trials over the past 20 years.

Chronic administration of beta3-AR agonists have demonstrated antidiabetic and antiobesity effects in obese and diabetic rodent models (2, 20, 23, 29, 37, 41, 42, 44), yet the interaction between lipid and glucose metabolism induced by beta3-AR agonists treatment is not clear: there were inconsistent effects on lipid levels, glucose and/or insulin levels, and insulin sensitivity after chronic administration of beta3-AR agonists (1, 39). Possible antidiabetic effects of beta3-AR agonists have been suggested by the observation of improved insulin-stimulated glucose disposal with enhanced insulin action in adipose tissues but with unchanged circulating glucose, insulin, and lipid levels in normal Sprague-Dawley rats after chronic activation of beta3-AR (14).

We have recently developed a type 2 diabetic mouse model by overexpressing a dominant negative IGF-I receptor specifically in skeletal muscle (MKR mice) (16). Hybrid formation of the mutated IGF-IR with the endogenous IGF-I and insulin receptors caused impaired insulin and IGF-I receptor signaling pathways in skeletal muscle of the MKR mice. This defect in skeletal muscle resulted in hyperinsulinemia, dyslipidemia, and beta-cell dysfunction leading eventually to type 2 diabetes, whereas total body fat and adipose tissue content were unchanged compared with normal wild-type (WT) mice. Whole body insulin resistance was associated with impaired insulin sensitivity in skeletal muscle as well as in adipose tissue and liver. A marked reduction of significantly elevated serum lipids [fatty acids (FA) and triglycerides (TG)] and liver lipid content following treatment with WY-14643, a peroxisome proliferator-activated receptor (PPAR){alpha} agonist, was associated with improvement in insulin sensitivity and diabetes in MKR mice (24). However, rosiglitazone, a PPAR{gamma} agonist, failed to improve insulin resistance and diabetic status of MKR mice despite a significant reduction in circulating lipid levels (25). Rosiglitazone increased glucose uptake by adipose tissue but had no effect on insulin sensitivity in muscle or liver (25). This lack of effect of the thiazolodinediones was associated with adiponectin resistance seen in these mice and may be causally related.

In this study, to elucidate the effects of beta3-AR agonists in a nonobese diabetic mouse model, we treated MKR mice with a highly selective beta3-AR agonist, CL-316243. Chronic beta3-AR activation reversed the whole body insulin insensitivity with enhanced insulin action on liver and adipose tissue and reduced hyperinsulinemia and hyperglycemia in MKR mice, whereas insulin resistance in skeletal muscle was not affected. This improvement was possibly associated with the beneficial effect of beta3-AR agonist on the lipid profiles and lipid metabolism.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Generation and characterization of MKR mice have been described before (16). Homozygous MKR male mice (FVB/N background) used for the current study were subjected to Southern blot analysis for genotyping, as previously described (16). FVB/N male mice were used as controls. Mice were maintained on a 12:12-h light-dark cycle, and all experiments were performed in agreement with National Institutes of Health guidelines and with the approval of the Animal Care and Use Committee of the National Institute of Diabetes and Digestive and Kidney Diseases.

Indirect calorimetry. Oxygen consumption and carbon dioxide production were measured using a four-chamber Oxymax system (Columbus Instruments, Columbus, OH) with one mouse/chamber and by testing transgenic mice simultaneously with littermate controls (17). Motor activity (total and ambulating) was determined by infrared beam interruption (Opto-Varimex mini, Columbus Instruments). Mice had free access to food and water. Resting O2 consumption was calculated as the average of the points with fewer than six ambulating beam breaks per minute and omission of the first hour of the experiment. The respiratory exchange ratio (RER; the ratio of CO2 produced to O2 consumed) was calculated using the same data points. Oxidation of carbohydrate produces an RER of 1.00, whereas FA oxidation results in an RER of ~0.70 (32). Results for O2 consumption were normalized to (body weight)0.75 (40).

The effect of the beta3-selective adrenergic agonist CL-316243 (6) on metabolic rate was measured as follows with each mouse serving as its own control. At ~9:00 AM, mice were placed into the calorimetry chambers (prewarmed to 30°C), and baseline data were collected. CL-316243 was injected (1 mg/kg ip) 3 h later, and after a 1-h delay data were collected for 3 h.

Acute administration of CL-316243. Six- to seven-week-old male WT and MKR mice were injected intraperitoneally either with CL-316243 (1 mg·kg–1·day–1) or with an equivalent volume of sterile saline. After 20 min, blood was collected from tail vein for serum FA levels.

Chronic administration of CL-316243. Six- to seven-week-old male WT and MKR mice were injected intraperitoneally with CL-316243 (1 mg·kg body wt–1·day–1) or with an equivalent volume of sterile saline daily (10:00 AM) for 3 wk. Body weight and food intake were measured daily or weekly, respectively. Blood was collected from the tail vein weekly between 11:00 AM and noon in nonfasting state. A glucometer (One Touch II, LifeScan) was used to measure glucose levels in the nonfasting state. Oxygen consumption was measured on day 20 for 24 h at 23°C. Mice were euthanized on day 21 of treatment after anesthetization using 2.5% Avertin (15–17 µl/g body wt) in the nonfasting state between 11:00 AM and noon. Tissues were collected for FA oxidation assay and immediately frozen in liquid nitrogen for measurement of tissue TG levels and RNA analysis.

Serum analysis. Serum was obtained from the tail vein between 11:00 AM and noon in the nonfasting state. Serum FA and TG levels were measured using a FA assay kit (Roche, Indianapolis, IN) and GPO-Trinder kit (Sigma, St. Louis, MO), respectively. Serum insulin, leptin, and adiponectin levels were determined using radioimmunoassay (Linco Research, St. Charles, MO).

Insulin and glucose tolerance tests. Insulin and glucose tolerance tests were done after 5–7 h of fasting. Mice were injected intraperitoneally with either insulin (0.5 U/kg body wt) or glucose (2 g/kg body wt). Blood glucose levels were determined from the tail vein at 0, 30, and 60 min after insulin injection and at 0, 15, 30, 60, and 120 min after glucose injection.

Hyperinsulinemic euglycemic clamp. The clamp studies were performed as developed by Kim et al. (26). Mice were treated with either CL-316243 or saline for 2 wk, as described above. On day 14 of treatment (4 days before the clamp experiment), mice were anesthetized with 100 mg/kg ketamine and 10 mg/kg xylazine. A catheter was inserted into a lateral incision on the right side of the neck and advanced into the superior vena cava via the right internal jugular vein. The catheter was then sutured into place according to the protocol of MacLeod and Shapiro (30). The day before the clamp analysis, mice received the final injection of CL-316243 or saline at 11:00 AM and were then fasted overnight. To conduct experiments in conscious animals with minimal stress, a tail restraint method was used during experiments. The basal rates of glucose turnover were measured by continuous infusion of [3-3H]glucose (0.02 µCi/min) for 120 min, which followed a bolus of 2.5 µCi, starting at 9:00 AM. Blood samples (20 µl) were taken at 90 and 115 min of the basal period for the determination of plasma [3H]glucose concentration. A 120-min hyperinsulinemic euglycemic clamp was started at 11:00 AM. Insulin was infused as a bolus of 150 mU/kg over a period of 3 min followed by continuous insulin infusion at the rate of 2.5 mU·kg–1·min–1 (Humulin R; Eli Lilly, Indianapolis, IN) to raise the plasma insulin concentration to ~2 ng/ml. During the clamp study, mice were restrained, and blood samples (20 µl) were collected via a small nick in the tail vein at 15-min intervals for the immediate measurement of plasma glucose concentration, and 20% glucose was infused at variable rates to maintain plasma glucose at ~160 mg/dl in WT mice (control MKR mice were clamped at ~240 mg/dl; because of severe insulin resistance, this was the lowest glucose level we could reach). Insulin-stimulated whole body glucose flux was estimated using a primed continuous infusion of high-pressure liquid chromatography-purified [3-3H]glucose (10-µCi bolus, 0.1 µCi/min; NEN Life Science Products, Boston, MA) throughout the clamps. To estimate insulin-stimulated glucose transport activity and metabolism in skeletal muscle, 2-deoxy-D-[1-14C]glucose (NEN Life Science Products) was administered as a bolus (10 µCi) at 45 min before the end of clamps. Blood samples (20 µl) were taken at 80, 85, 90, 100, 110, and 120 min after the start of clamps for the determination of plasma [3H]glucose, 2-deoxy-D-[1-14C]glucose, and 3H2O concentrations. Additional blood samples (10 µl) were collected before the start and at the end of clamp studies for measurements of plasma insulin concentration. All infusions were performed using microdialysis pumps (CMA/Microdialysis, Acton, MA). At the end of the clamp period, animals were anesthetized with ketamine-xylazine injection. Within 5 min, gastrocnemius muscle from hindlimbs, epididymal and brown adipose tissue, and liver were removed. Each tissue, once exposed, was dissected within 2 s, frozen immediately using liquid nitrogen-cooled aluminum blocks, and stored at –70°C for later analysis.

Tissue TG content determination. Livers were powdered, and tissue TG were extracted in chloroform-methanol solution. The solution was centrifuged after addition of 2% KH2PO4, and the lower phase was collected for evaporation. Then, isopropyl alcohol was added to dissolve the remaining pellet. The amount of released glycerol was measured by radiometric assay as described before (7, 9).

Body fat content determination. Body composition was measured in nonanesthetized mice by use of the Bruker minispec NMR analyzer mq10 (Bruker Optics, Woodlands, TX).

Food intake. Mice were caged individually and treated with either CL-316243 or vehicle (sterile saline), as described above. The amount of food in the feeding container was measured at days 0 and 10 of treatment. Food intake was normalized to the body weight of each mouse and was expressed as grams of food per gram of body weight0.75 per day.

TG secretion. TG secretion was determined by the measurement of the increased circulating TG after lipase inhibition by WR-1339 (Sigma), as described previously (13). At day 21 of treatment with either CL-316243 or saline, mice were given ad libitum access to a fat-free diet (Frosted Flakes; Kellogg, Battle Creek, MI) for 4 h. Mice were then anesthetized with ketamine (100 mg/kg; Fort Dodge Animal Health, Fort Dodge, IA) and xylazine (10 mg/kg; Phoemx Scientific, St. Joseph, MO). WR-1339 (100 µl from a solution of 1:10 dilution in phosphate-buffered saline) was injected into the tail vein, and blood samples were collected at 0, 1, and 2 h. Results were expressed as milligrams of TG per kilogram body weight per hour, and plasma volume was presumed as a 3.5% of body weight.

TG clearance test. TG clearance from the circulation in 4-h-fasted mice was measured after gavage of 400 µl of peanut oil after 3 wk of treatment. Blood samples were withdrawn hourly from the tail vein for 6 h.

FA oxidation assay. Isolated free adipocytes from epididymal adipose tissue (36) of mice, after 3 wk of treatment, were incubated with BSA-bound palmitic acid (4.3 µmol/l) and [3H]palmitic acid [9,10-3H(N), 55.6 pmol/l; PerkinElmer Life Sciences, Boston, MA] in 5 mM glucose, Krebs-Ringer-HEPES-albumin buffer (pH 7.4, 20 mg/ml FA-free BSA) buffer at 37°C for 0, 30, and 60 min, respectively. At every time point, the cell suspension was immediately added into a microtube, which contained the same volume of mineral oil, and was then briefly centrifuged. The lower phase was added into a column filled with 1 ml of resin (Bio-Rad AG1-X8, 200–400 mesh), which retains FAs. The column was washed with water, and the eluate was counted for 3H2O production. Adipocyte DNA content was measured by fluorometric quantification assay (15) using bis-benzamide as a dye and calf thymus high polymerized DNA (Sigma) as a standard. The results were expressed as the value of oxidized palmitic acid (pmol) normalized by adipocyte DNA (µg). The stock solutions of palmitic acid bound to FA-free BSA were prepared as described previously (8), and the nonesterified fatty acid concentration was verified using a NEFA C kit (Wako Chemicals, Richmond, VA).

FA oxidation on isolated soleus muscle. All experiments were performed in Krebs-Henseleit buffer (KHB; in mM: 118.5 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 2.5 CaCl2, 0.5 EDTA, and 25 NaHCO3, supplemented with 5 mM D-glucose) (22). All BSA and palmitate-BSA complexes used were dialyzed extensively against KHB, and all buffers were continuously gassed with 95% O2-5% CO2. Soleus muscles, including tendons, were rapidly and carefully excised from 12-wk-old animals and placed in capped 10-ml vials containing 5 ml of KHB supplemented with 2% BSA. After a 20-min incubation in a shaking water bath (150 strokes/min), muscles were incubated with 0.2 mM palmitate and 0.45 µCi/ml [1-14C]palmitate (ICN, Irvine, CA) complexed to 2% BSA. Antibiotic solution (Sigma) was added to prevent bacterial growth. Incubations were carried out at 30°C for 30 min in vials sealed with rubber stoppers (Kontes, Vineland, NJ) to maintain the interior of the vial at 95% O2-5% CO2. The incubation medium was quickly transferred to new capped vials equipped with a center well containing 200 µl of ethanolamine-ethylene glycol (1:2 vol/vol). Perchloric acid was added through the cap to a final concentration of 0.6 M, and vials were incubated overnight with gentle shaking. The amount of CO2 produced was determined by liquid scintillation counting of the ethanolamine-ethylene glycol mixture.

RNA analysis. Total RNA from liver, WAT, and BAT was isolated using TRIzol reagent (Life Technologies, Rockville, MD), and Northern blot analysis was performed as previously described. (23)

Histology. The left gonadal fat pad was removed following euthanasia and fixed overnight in 4% paraformaldehyde in PBS. The tissues were then transferred to 70% ethanol and embedded in paraffin. Samples were cut into 5-µm sections, and hematoxylin-eosin staining was performed. Immunostaining for cytochrome-c oxidase was performed using an antibody from Molecular Probes (Eugene, OR).

Statistical analysis. All data are expressed as means ± SE. Student's t-test (unpaired and paired) was used to determine statistically significant differences between genotype.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Acute effect of CL-316243 on MKR mice. To determine whether beta3-adrenergic responsiveness is intact in MKR mice, we measured serum FAs and metabolic rate in WT and MKR mice after acute beta3-adrenergic stimulation. Serum FAs rose four- to sixfold in both WT and MKR mice, indicating an increase in the lipolysis from BAT and WAT (Fig. 1A). In response to acute CL-316243 treatment, both WT and MKR mice increased O2 consumption by ~100% and decreased the RER (ratio of CO2 produced to O2 consumed), an indicator of metabolic fuel source, suggesting increased net FA oxidation. Therefore, these data demonstrate that intact beta3-adrenergic responses are preserved in MKR mice.


Figure 1
View larger version (11K):
[in this window]
[in a new window]
  		Fig. 1. Acute effect of CL-316243 (CL) on serum fatty acid (FA) levels (A), resting O2 consumption (B), and respiratory exchange ratio (C) in wild-type (WT) and MKR mice. Six-week-old MKR and WT mice were injected with saline ({blacksquare}) or CL-316243 ({square}) (1 mg/kg ip), and 20 min after injections blood was collected for serum FA levels. Mice were prewarmed at 30°C, and after 3 h CL-316243 was injected (1 mg/kg ip). One hour later, resting O2 consumption and RER levels were measured over a 2-h period, as described in MATERIALS AND METHODS. Results are expressed as means ± SE; n = 6–8/group. *Differences between treated and untreated group were significant at P < 0.05.

 
Effect of chronic CL-316243 treatments on body composition. To explore potential antidiabetic effects of beta3-adrenergic stimulation in MKR mice, we treated mice with CL-316243 for 3 wk; there was no effect on body weight. However, total fat content decreased significantly in both WT and MKR mice (25 and 16%, respectively; Table 1). Interestingly, various fat depots were affected differently: epididymal pad weight was decreased by 33 and 50% in WT and MKR mice, respectively; however, inguinal fat weight did not change after 3 wk of treatment with CL-316243 (Table 1). BAT did not change after CL-316243 treatment in WT mice [194 ± 27 vs. 140 ± 15 mg, P = not sigificant (NS)] or in MKR mice (120 ± 3 vs.138 ± 11 mg, P = NS).


View this table:
[in this window]
[in a new window]
  		Table 1. Oxygen consumption and total body fat content

 
As expected, chronic CL-316243 treatment caused significant increases in O2 consumption. Average 24-h O2 consumption measured at ambient temperature (23°C) increased by 12 and 11% in MKR and WT mice, respectively (Table 1). This increase in O2 consumption was correlated with a slight, probably compensatory, increase in food intake of both WT and MKR mice (14 and 10%, respectively; Table 1). This may explain why both WT and MKR mice maintained body weight and showed only modest reductions in total fat mass despite a chronic increase in metabolic rate.

Treatment caused a reduction in the serum leptin levels by 25 and 15% in WT and MKR mice, presumably due to decreased body fat content (Table 1). Serum adiponectin level in WT mice was significantly increased, but not in MKR mice, in response to CL-316243 treatment (Table 1).

Antidiabetic effect of chronic CL-316243 treatment on MKR mice. beta3-Adrenergic agonist CL-316243 treatment of MKR mice for 3 wk caused a marked improvement in hyperglycemia and hyperinsulinemia. Blood glucose levels fell from 242 ± 28.4 to 125 ± 3.1 mg/dl (Fig. 2A), whereas serum insulin levels fell from the hyperinsulinemic range of 21 ± 3.3 to 4 ± 2.4 ng/ml (Fig. 2B). In contrast, chronic CL-316243 treatments did not alter the levels of these parameters in WT mice.


Figure 2
View larger version (13K):
[in this window]
[in a new window]
  		Fig. 2. Effect of CL-316243 on blood glucose (A), serum insulin (B), insulin tolerance (C), glucose tolerance (D), and area under the curve (AUC) from glucose tolerance tests (E) in WT and MKR mice. Six- to seven-week-old MKR and WT mice were injected daily with saline ({blacksquare}) or CL-316243 ({square}) (1 mg/kg ip) for 3 wk. In the nonfasting state, blood or serum was collected, and under fasting state insulin and glucose tolerance tests were performed. Values are expressed as means ± SE; n = 6–9/group. #P < 0.05 vs. WT; *P < 0.05 between treated and untreated group.

 
Insulin tolerance tests were performed after 3 wk of CL-316243 treatment. Consistent with previous studies (16, 24) (25), MKR mice exhibited severe insulin insensitivity compared with WT mice (Fig. 2C). Chronic CL-316243 treatment in MKR mice significantly enhanced the ability of insulin to clear blood glucose at 60 min from 88 to 63% of initial levels (to the levels of WT mice), indicating enhanced insulin sensitivity (Fig. 2C). A slight increase in insulin sensitivity was observed in WT mice after CL-316243 treatment (Fig. 2C).

Consistent with our earlier studies (16, 25), control MKR mice had higher basal fasting glucose levels compared with WT mice and were glucose intolerant (Fig. 2D). Glucose tolerance was markedly improved in MKR mice after treatment (Fig. 2D). The area under curve (AUC) was significantly reduced by 63% in MKR mice after CL-316243 treatment compared with saline-treated MKR mice (Fig. 2E). CL-316243 treatment caused a significant decrease in AUC of WT mice by 20% (Fig. 2E).

Taken together, these data demonstrate that chronic beta3-adrenergic agonist treatment significantly improved insulin insensitivity and glucose homeostasis in MKR mice.

CL-316243 treatment increased insulin sensitivity in hepatic and adipose tissue in MKR mice. MKR mice exhibit insulin resistance in all major insulin responsive tissues: muscle, liver, and fat (16). To assess the mechanism of the insulin-sensitizing effect of CL-316243, we performed hyperinsulinemic euglycemic clamp studies. After an overnight fast, CL-316243-treated MKR mice showed a significant reduction in basal serum insulin levels to the level of WT mice (from 2.4 ± 1.3 to 0.5 ± 0.1 ng/ml) despite unchanged hyperglycemia. The glucose infusion rate required to maintain euglycemia was significantly higher after CL-316243 treatment in both MKR and WT mice, suggesting improvement in whole body insulin sensitivity. In MKR mice, basal endogenous glucose production (EGP) was significantly higher than in WT controls; CL-316243 treatment lowered it to the WT levels (Table 2). During the clamp, CL-316243-treated MKR mice revealed a further significant reduction (~67%) in EGP compared with saline-treated MKR mice (Fig. 3B). There was a significant reduction in clamp EGP in WT mice after treatment compared with saline-treated WT mice (Fig. 3B). Glycogen synthesis rate in liver was significantly increased in MKR mice after CL-316243 treatment, and a similar nonsignificant trend was seen in WT mice after CL-316243 treatment (Table 2). These data suggest that CL-316243 treatment enhanced hepatic insulin sensitivity in both WT and MKR mice. However, the values for whole body glucose uptake and muscle glucose uptake were not increased in either WT or MKR mice after Cl316,243 treatment (Fig. 3, C and D). In WT mice, glucose uptake in WAT and BAT did not change after CL-316243 treatment, whereas in MKR mice it was significantly increased (Fig. 3, E and F), indicating enhanced adipose tissue insulin sensitivity in MKR mice after CL-316243 treatment. GLUT4 mRNA tended to increase in WAT after CL-316243 treatment in WT (1.95 ± 0.49 vs. 3.32 ± 1.14 AU) and MKR (2.74 ± 0.38 vs. 6.20 ± 2.33 AU) mice, whereas no change was observed in BAT in both groups of mice (data not shown). The hyperinsulinemic euglycemic clamp analysis showed a significantly higher muscle glucose uptake and unchanged whole body glucose uptake in MKR mice compared with WT mice. Certain limitations in the hyperinsulinemic euglycemic clamp technique may explain the data. These observations are probably due to significantly increased glucose levels during clamps in MKR mice compared with WT mice. This increased glucose uptake is most likely independent of the insulin-stimulated pathway in MKR mice.


View this table:
[in this window]
[in a new window]
  		Table 2. Effects of CL-316243 treatment on metabolic parameters during basal and hyperinsulinemic euglycemic clamp

 

Figure 3
View larger version (14K):
[in this window]
[in a new window]
  		Fig. 3. Effect of CL-316243 on glucose infusion rate (A), clamp endogenous glucose production (EGP; B), whole body glucose uptake (C), muscle glucose uptake (D), white adipose tissue (WAT) glucose uptake (E), and brown adipose tissue (BAT) glucose uptake (F) during hyperinsulinemic euglycemic clamp analysis. Six-week-old MKR and WT mice were injected with saline ({blacksquare}) or CL-316243 ({square}) (1 mg/kg ip) for 3 wk. After 12 h of fasting, mice were subjected to hyperinsulinemic euglycemic clamp. Data are expressed as means ± SE; n = 8–9/group. *P < 0.05 vs. untreated group within genotype; #P < 0.05 vs. WT.

 
Chronic CL-316243 treatment improves lipid metabolism in MKR mice. We next studied the effects of chronic CL-316243 treatment on lipid metabolism in WT and MKR mice. Treating MKR mice with CL-316243 for 3 wk significantly lowered serum and hepatic lipid levels. Serum TG levels fell from the hypertriglyceridemic range of 148 ± 14.7 to 60 ± 21.3 mg/dl in MKR mice after treatment (Fig. 4A), whereas serum FA levels were reduced from 0.4 ± 0.03 to 0.2 ± 0.03 mM (Fig. 4B). CL-316243 treatment significantly decreased liver TG content by ~38% in MKR mice (Fig. 4C). The decreased liver TG levels correlated well with a reduction in in vivo TG secretion rate (by ~37%) in MKR mice after treatment (Fig. 4D). However, treatment did not change circulating and liver TG and FA levels in WT mice (Fig. 4). The expressions in liver of fatty acid synthase (FAS), stearoyl-CoA desaturase (SCD)-1, glycogen synthase, glucokinase (GK), phosphoenolpyruvate carboxykinase (PEPCK), and PPAR{alpha} mRNA remained unchanged in both genotypes after treatment (data not shown).


Figure 4
View larger version (17K):
[in this window]
[in a new window]
  		Fig. 4. Effect of CL-316243 on serum triglyceride (TG; A), serum fatty acids (FAs; B), liver TG content (C), TG secretion rate after WR-1339 injection (D), TG clearance after peanut oil gavage (E), and normal uptake of intestinal lipid (measured as increase in plasma TG levels in mg/dl) after TG clearance with peanut oil load was inhibited by WR-1339 injection (F) in WT and MKR mice. Six- to seven-week-old MKR and WT mice were injected daily with saline ({blacksquare}) or CL-316243 ({square}) (1 mg/kg ip) for 3 wk. In the nonfasting state, serum and liver were collected. Data are expressed as means ± SE; n = 6–9/group. *P < 0.05 between treated and untreated group.

 
To determine whether the reduction of serum TG level in MKR mice after CL-316243 treatment is associated with enhanced TG clearance, we measured serum TG levels after an oral lipid load. At 3 h, serum TG peaked at 677 and 805 mg/dl in WT and MKR mice, respectively, and then fell gradually (Fig. 4E). CL-316243 treatment lowered the peak of serum TG levels to 184 mg/dl in MKR mice and 459 mg/dl in WT mice 2 h after the lipid load, and serum TG were reduced rapidly, indicating more efficient TG clearance in MKR mice after treatment (Fig. 4E). This was not due to poor TG uptake from the intestine, because, in the absence of TG clearance [caused by WR-1339, a lipoprotein lipase (LPL) inhibitor], serum TG increased equally in control and CL-316243-treated mice after oral TG load (Fig. 4F). Taken together, these data indicate that CL-316243 treatment accelerated TG clearance in both WT and MKR mice.

Chronic CL-316243 treatment increases adipose tissue FA oxidation in MKR mice. It is presumed that beta3-adrenergic stimulation increases muscle (2) and adipose tissue FA oxidation, thus leading to rapid reduction of circulating TG levels and hepatic TG content. The level of muscle FA oxidation was significantly decreased by 39% in MKR mice after treatment but was not altered in WT mice after treatment (Fig. 5A). We next measured FA oxidation in the isolated epididymal adipose tissue following chronic treatment. CL-316243 treatment caused a marked increase (fourfold) in adipose tissue FA oxidation in MKR mice, but only a onefold increase in WT mice (Fig. 5B). LPL mRNA levels showed a tendency to increase in WAT in both WT (1.63 ± 0.01 vs. 3.72 ± 0.46 AU) and MKR (5.06 ± 1.2 vs. 6.33 ± 0.88 AU) mice, whereas in BAT it tended to increase only in MKR mice (2.62 ± 1.36 vs. 4.05 ± 1 AU). Therefore, these data suggest that adipose tissue, rather than muscle, is primarily involved in the reduced lipid levels in MKR mice after chronic beta3-adrenergic agonist treatment.


Figure 5
View larger version (59K):
[in this window]
[in a new window]
  		Fig. 5. Effects of CL-316243 on FA oxidation and morphology of adipose tissues. A: FA oxidation by soleus muscle. Palmitic acid (PA) oxidation rates in isolated soleus muscle were determined at an FA-to-BSA ratio of 0.67 (n = 6–8/group). B: palmitic acid oxidation by epididymal adipose tissues. Isolated adipocytes were incubated with BSA-bound palmitic acid (4.33 µmol/l) for 0, 30, and 60 min (n = 3/group). FA oxidation was measured as described in METHODS AND MATERIALS. C: morphology of epididymal adipose tissue from hematoxylin-eosin staining. D: immunostaining for presence of cytochrome-c oxidase in both WT and MKR epididymal adipose tissue before and after treatment with CL-316243. Six- to seven-week-old MKR and WT mice were injected with saline ({blacksquare}) or CL-316243 ({square}) (1 mg/kg ip) for 3 wk. Muscle and epididymal adipose tissue were removed to measure FA oxidation or to process morphology study in the nonfasting state. Data are expressed as means ± SE. *Differences between treated and untreated were significant at P < 0.05.

 
Several studies have reported that beta3-adrenergic agonists decrease the size of adipocytes in epididymal adipose tissue (10, 18, 20). Concomitant changes in mitochondrial content and cytochrome-c oxidase expression in epididymal or inguinal adipose tissue have also been reported (23). Hematoxylin-eosin staining in epididymal adipose tissue showed typical unilocular cells in both WT and MKR mice (Fig. 5C, left). As expected, treatment with CL-316243 caused the formation of small adipocytes and mutilocular adipocytes in epididymal adipose tissues of MKR mice (Fig. 5C, right). Accordingly, immunostaining for cytochrome-c oxidase showed an increase in both WT and MKR epididymal adipose tissue (Fig. 5D). Compared with MKR mice, the effects of CL-316243 on morphology changes and cytochrome-c oxidase content in epididymal adipose tissue observed in WT mice after treatment were less marked (Fig. 5, C and D, left).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study demonstrated that chronic treatment with a beta3-adrenergic agonist, such as CL-316243, markedly improved hyperglycemia and hyperinsulinemia concurrently with significantly improved lipid profiles (circulating and tissue lipid contents) and metabolism in MKR mice that are diabetic but not obese. This improvement was paralleled by enhanced whole body insulin sensitivity, which was associated with increased hepatic insulin responsiveness and insulin-stimulated glucose uptake in adipose tissue. These results suggest that potentiation of insulin action on liver and adipose tissues plays a crucial role in the antidiabetic effects of beta3-adrenergic agonists in MKR mice that are genetically defective in muscle insulin action.

The importance of hepatic insulin action for whole body insulin sensitivity and glucose homeostasis in MKR mice has been shown previously (24, 25). An increase in hepatic lipogenesis, based on enhanced hepatic expression of PPAR{gamma} and SCD-1 mRNA, was associated with hepatic insulin resistance (25). Enhanced hepatic insulin action was correlated with a reduction in hepatic TG levels after treatment of WY-14643, a PPAR{alpha} agonist (24). Consistently, reduction in hepatic TG levels, paralleled with a decrease in TG secretion rate, was related to the improvement in hepatic insulin sensitivity in MKR mice after chronic CL-316243 treatment. This improved hepatic insulin sensitivity was shown by reduced EGP and increased hepatic glycogen synthesis rate in response to insulin during the hyperinsulinemic euglycemic clamp. Due to exclusive and abundant expression of beta3-AR in adipose tissue, this effect of CL-316243 on hepatic insulin sensitivity could be secondary to the increased fatty acid utilization in adipose tissues, resulting in reduced hepatic lipid contents. In fact, CL-316243 treatment did not affect the expression of hepatic mRNA levels such as FAS, SCD-1, glycogen synthase, and PEPCK mRNA (data not shown).

The mechanisms responsible for enhanced insulin sensitivity in adipose tissues after activation of beta3-ARs include the appearance of small adipocytes, increased expression of uncoupling protein-1 in both BAT and WAT (23, 35), the expression of insulin receptors and GLUT4, increased postreceptor activity, increased mitochondrial content(23), cytochrome-c oxidase content (23), and impaired adipogenesis by downregulation of PPAR{gamma} and aP2 gene (23, 31). Chronic treatment with the beta3-adrenergic agonist in MKR mice caused an appearance of small adipocytes in both inguinal and epididymal adipose tissues, whereas there was a lesser effect in WT mice. Furthermore, the beta3-adrenergic agonist induced an improvement in insulin sensitivity in adipose tissues as well as in liver, leading to an increase in whole body insulin sensitivity and glucose metabolism in MKR mice. Improved insulin action in adipose tissues, but not in skeletal muscle, was responsible for the enhanced whole body insulin-stimulated glucose disposal in nonobese rats after beta3-AR activation (14). These results indicate that adipose tissue is a possible primary target site for the antidiabetic effect of the beta3-adrenergic agonist.

The expression levels of beta3-AR are adipose tissue depot dependent. For example, in humans, adipocytes derived from intra-abdominal or visceral depots reveal more beta3-AR with better responsiveness to beta3-adrenergic agonists, whereas adipocytes derived from subcutaneous depots have few beta3-ARs. Consistently, there was a greater reduction in epididymal adipose tissue weight than in inguinal adipose tissue weight in both WT and MKR mice after CL-316243 treatments. This reduction of epididymal adipose tissue weight as well as total body fat content in both WT and MKR mice indicate a similar antiobesity effectiveness of beta3-adrenergic agonist on both WT and MKR mice.

It is presumed that enhanced expression level of LPL and appearance of lipid in BAT result in increased TG clearance with chronic activation of beta3-AR (10, 18, 20, 33). Chronic administration of CL-316243 in MKR mice markedly enhanced TG clearance, whereas there was a lesser effect in WT mice. Consistently, morphological changes in epididymal adipose tissue such as formation of mutilocular adipocytes, a possible indicator of reappearance of BAT, were less obvious in WT mice after treatment compared with treated MKR mice. Therefore, the effectiveness of beta3-adrenergic agonist on WAT by appearance of multilocular adipocytes, in part, contribute to the increase in TG clearance.

Stimulation of FA oxidation as well as increased lipolysis in WAT is associated with the reduction in circulating FA levels after chronic activation of beta3-AR (5, 31, 43). Our findings demonstrated increased lipolysis in an acute response to CL-316243 treatments and enhanced FA oxidation in adipocytes of MKR mice after chronic CL-316243 treatment. However, FA oxidation in gastrocnemius muscle was not increased in MKR mice after chronic CL-316243 treatment, possibly related to less availability of FAs by a reduction of circulating FA levels after chronic activation of beta3-AR and exclusively high expression of beta3-AR in adipose tissues. It has been reported that muscle LPL activity was not affected with activation of beta3-AR (33). These data indicate the significant role of adipose tissue in the reduction of circulating FA levels after chronic activation of beta3-AR.

In summary, intact beta3-adrenergic responses on beta3-adrenergic activation with CL-316243 treatment are retained in diabetic MKR mice that have impaired insulin and IGF-I signaling pathways in skeletal muscle. Chronic administration of CL-316243 significantly improves whole body insulin sensitivity and glucose homeostasis in MKR mice, mainly due to enhanced insulin action on liver and adipose tissues. This enhanced insulin sensitivity was associated with the beneficial effect of the beta3-adrenergic agonist CL-316243 on lipid metabolism in MKR mice. Reduction in circulating and hepatic lipid levels was, in part, related to increased TG clearance, decreased TG secretion rate, and increased adipocyte FA oxidation in MKR mice in response to CL-316243 treatment. Increased metabolic rate paralleled the reduction in epididymal adipose tissue and total body fat content in MKR mice after CL-316243 treatment.

In conclusion, enhanced insulin action on liver and adipose tissue, mainly due to increased adipocyte FA oxidation, with beta3-AR activation is able to counterbalance the diabetes state of MKR mice that are genetically defective in IGF-I/insulin receptor signaling pathways of skeletal muscle.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by federal funds from the Intramural Research Program, Diabetes Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, and a research grant to D. LeRoith from the American Diabetes Association.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. LeRoith, Division of Endocrinology, Diabetes, and Bone Diseases, Mt. Sinai School of Medicine, 1 Gustave Levy Place (1055), New York, NY 10029 (e-mail: derek.leroith{at}mssu.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.

* These authors contributed equally to this study. Back


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Arbeeny CM, Meyers DS, Hillyer DE, and Bergquist KE. Metabolic alterations associated with the antidiabetic effect of beta 3-adrenergic receptor agonists in obese mice. Am J Physiol Endocrinol Metab 268: E678–E684, 1995.[Abstract/Free Full Text]
  2. Arch JR. Beta(3)-adrenoceptor agonists: potential, pitfalls and progress. Eur J Pharmacol 440: 99–107, 2002.[CrossRef][ISI][Medline]
  3. Arch JR and Kaumann AJ. Beta 3 and atypical beta-adrenoceptors. Med Res Rev 13: 663–729, 1993.[CrossRef][ISI][Medline]
  4. Berkowitz DE, Nardone NA, Smiley RM, Price DT, Kreutter DK, Fremeau RT, and Schwinn DA. Distribution of beta 3-adrenoceptor mRNA in human tissues. Eur J Pharmacol 289: 223–228, 1995.[CrossRef][ISI][Medline]
  5. Berraondo B, Marti A, Duncan JS, Trayhurn P, and Martinez JA. Up-regulation of muscle UCP2 gene expression by a new beta3-adrenoceptor agonist, trecadrine, in obese (cafeteria) rodents, but down-regulation in lean animals. Int J Obes Relat Metab Disord 24: 156–163, 2000.[CrossRef][ISI][Medline]
  6. Bloom JD, Dutia MD, Johnson BD, Wissner A, Burns MG, Largis EE, Dolan JA, and Claus TH. Disodium (R,R)-5-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]propyl]-1,3-benzodioxole-2,2-dicarboxylate (CL 316,243). A potent beta-adrenergic agonist virtually specific for beta 3 receptors A promising antidiabetic and antiobesity agent. J Med Chem 35: 3081–3084, 1992.[CrossRef][ISI][Medline]
  7. Bradley DC and Kaslow HR. Radiometric assays for glycerol, glucose, and glycogen. Anal Biochem 180: 11–16, 1989.[CrossRef][ISI][Medline]
  8. Brun T, Assimacopoulos-Jeannet F, Corkey BE, and Prentki M. Long-chain fatty acids inhibit acetyl-CoA carboxylase gene expression in the pancreatic beta-cell line INS-1. Diabetes 46: 393–400, 1997.[Abstract]
  9. Burant CF, Sreenan S, Hirano K, Tai TA, Lohmiller J, Lukens J, Davidson NO, Ross S, and Graves RA. Troglitazone action is independent of adipose tissue. J Clin Invest 100: 2900–2908, 1997.[ISI][Medline]
  10. Charon C, Dupuy F, Marie V, and Bazin R. Effect of the beta-adrenoceptor agonist BRL-35135 on development of obesity in suckling Zucker (fa/fa) rats. Am J Physiol Endocrinol Metab 268: E1039–E1045, 1995.[Abstract/Free Full Text]
  11. Collins S, Cao W, and Robidoux J. Learning new tricks from old dogs: beta-adrenergic receptors teach new lessons on firing up adipose tissue metabolism. Mol Endocrinol 18: 2123–2131, 2004.[Abstract/Free Full Text]
  12. Collins S, Daniel KW, Rohlfs EM, Ramkumar V, Taylor IL, and Gettys TW. Impaired expression and functional activity of the beta 3- and beta 1-adrenergic receptors in adipose tissue of congenitally obese (C57BL/6J ob/ob) mice. Mol Endocrinol 8: 518–527, 1994.[Abstract]
  13. Colombo C, Haluzik M, Cutson JJ, Dietz KR, Marcus-Samuels B, Vinson C, Gavrilova O, and Reitman ML. Opposite effects of background genotype on muscle and liver insulin sensitivity of lipoatrophic mice. Role of triglyceride clearance. J Biol Chem 278: 3992–3999, 2003.[Abstract/Free Full Text]
  14. de Souza CJ, Hirshman MF, and Horton ES. CL-316,243, a beta3-specific adrenoceptor agonist, enhances insulin-stimulated glucose disposal in nonobese rats. Diabetes 46: 1257–1263, 1997.[Abstract]
  15. Downs TR and Wilfinger WW. Fluorometric quantification of DNA in cells and tissue. Anal Biochem 131: 538–547, 1983.[CrossRef][ISI][Medline]
  16. Fernandez AM, Kim JK, Yakar S, Dupont J, Hernandez-Sanchez C, Castle AL, Filmore J, Shulman GI, and Le Roith D. Functional inactivation of the IGF-I and insulin receptors in skeletal muscle causes type 2 diabetes. Genes Dev 15: 1926–1934, 2001.[Abstract/Free Full Text]
  17. Gavrilova O, Marcus-Samuels B, and Reitman ML. Lack of responses to a beta3-adrenergic agonist in lipoatrophic A-ZIP/F-1 mice. Diabetes 49: 1910–1916, 2000.[Abstract]
  18. Ghorbani M, Claus TH, and Himms-Hagen J. Hypertrophy of brown adipocytes in brown and white adipose tissues and reversal of diet-induced obesity in rats treated with a beta3-adrenoceptor agonist. Biochem Pharmacol 54: 121–131, 1997.[CrossRef][ISI][Medline]
  19. Grujic D, Susulic VS, Harper ME, Himms-Hagen J, Cunningham BA, Corkey BE, and Lowell BB. Beta3-adrenergic receptors on white and brown adipocytes mediate beta3-selective agonist-induced effects on energy expenditure, insulin secretion, and food intake. A study using transgenic and gene knockout mice. J Biol Chem 272: 17686–17693, 1997.[Abstract/Free Full Text]
  20. Himms-Hagen J, Cui J, Danforth E Jr, Taatjes DJ, Lang SS, Waters BL, and Claus TH. Effect of CL-316,243, a thermogenic beta3-agonist, on energy balance and brown and white adipose tissues in rats. Am J Physiol Regul Integr Comp Physiol 266: R1371–R1382, 1994.[Abstract/Free Full Text]
  21. Hoffstedt J, Poirier O, Thorne A, Lonnqvist F, Herrmann SM, Cambien F, and Arner P. Polymorphism of the human beta3-adrenoceptor gene forms a well-conserved haplotype that is associated with moderate obesity and altered receptor function. Diabetes 48: 203–205, 1999.[ISI][Medline]
  22. Ibrahimi A, Bonen A, Blinn WD, Hajri T, Li X, Zhong K, Cameron R, and Abumrad NA. Muscle-specific overexpression of FAT/CD36 enhances fatty acid oxidation by contracting muscle, reduces plasma triglycerides and fatty acids, and increases plasma glucose and insulin. J Biol Chem 274: 26761–26766, 1999.[Abstract/Free Full Text]
  23. Kato H, Ohue M, Kato K, Nomura A, Toyosawa K, Furutani Y, Kimura S, and Kadowaki T. Mechanism of amelioration of insulin resistance by beta3-adrenoceptor agonist AJ-9677 in the KK-Ay/Ta diabetic obese mouse model. Diabetes 50: 113–122, 2001.[Abstract/Free Full Text]
  24. Kim H, Haluzik M, Asghar Z, Yau D, Joseph JW, Fernandez AM, Reitman ML, Yakar S, Stannard B, Heron-Milhavet L, Wheeler MB, and LeRoith D. Peroxisome proliferator-activated receptor-alpha agonist treatment in a transgenic model of type 2 diabetes reverses the lipotoxic state and improves glucose homeostasis. Diabetes 52: 1770–1778, 2003.[Abstract/Free Full Text]
  25. Kim H, Haluzik M, Gavrilova O, Yakar S, Portas J, Sun H, Pajvani UB, Scherer PE, and LeRoith D. Thiazolidinediones improve insulin sensitivity in adipose tissue and reduce the hyperlipidaemia without affecting the hyperglycaemia in a transgenic model of type 2 diabetes. Diabetologia 47: 2215–2225, 2004.[CrossRef][ISI][Medline]
  26. Kim JK, Michael MD, Previs SF, Peroni OD, Mauvais-Jarvis F, Neschen S, Kahn BB, Kahn CR, and Shulman GI. Redistribution of substrates to adipose tissue promotes obesity in mice with selective insulin resistance in muscle. J Clin Invest 105: 1791–1797, 2000.[ISI][Medline]
  27. Krief S, Lonnqvist F, Raimbault S, Baude B, Van Spronsen A, Arner P, Strosberg AD, Ricquier D, and Emorine LJ. Tissue distribution of beta 3-adrenergic receptor mRNA in man. J Clin Invest 91: 344–349, 1993.[ISI][Medline]
  28. Liu YL and Stock MJ. Acute effects of the beta 3-adrenoceptor agonist, BRL 35135, on tissue glucose utilisation. Br J Pharmacol 114: 888–894, 1995.[ISI]
  29. Lowell BB and Flier JS. The potential significance of beta 3 adrenergic receptors. J Clin Invest 95: 923, 1995.[ISI][Medline]
  30. MacLeod JN and Shapiro BH. Repetitive blood sampling in unrestrained and unstressed mice using a chronic indwelling right atrial catheterization apparatus. Lab Anim Sci 38: 603–608, 1988.[ISI][Medline]
  31. Margareto J, Larrarte E, Marti A, and Martinez JA. Up-regulation of a thermogenesis-related gene (UCP1) and down-regulation of PPARgamma and aP2 genes in adipose tissue: possible features of the antiobesity effects of a beta3-adrenergic agonist. Biochem Pharmacol 61: 1471–1478, 2001.[CrossRef][ISI][Medline]
  32. McLean JATG. Animal and Human Calorimetry. Cambridge, UK: Cambridge Univ. Press, 1987.
  33. Milagro FI and Martinez JA. Effects of the oral administration of a beta3-adrenergic agonist on lipid metabolism in alloxan-diabetic rats. J Pharm Pharmacol 52: 851–856, 2000.[CrossRef][ISI][Medline]
  34. Nagase I, Yoshida T, Kumamoto K, Umekawa T, Sakane N, Nikami H, Kawada T, and Saito M. Expression of uncoupling protein in skeletal muscle and white fat of obese mice treated with thermogenic beta 3-adrenergic agonist. J Clin Invest 97: 2898–2904, 1996.[ISI][Medline]
  35. Oana F, Homma T, Takeda H, Matsuzawa A, Akahane S, Isaji M, and Akahane M. DNA microarray analysis of white adipose tissue from obese (fa/fa) Zucker rats treated with a beta3-adrenoceptor agonist, KTO-7924. Pharmacol Res 52: 395–400, 2005.[CrossRef][ISI][Medline]
  36. Rodbell M. Metabolism of isolated fat cells. I. Effects of hormones on glucose metabolism and lipolysis. J Biol Chem 239: 375–380, 1964.[Free Full Text]
  37. Smith SA, Levy AL, Sennitt MV, Simson DL, and Cawthorne MA. Effects of BRL 26830, a novel beta-adrenoceptor agonist, on glucose tolerance, insulin sensitivity and glucose turnover in Zucker (fa/fa) rats. Biochem Pharmacol 34: 2425–2429, 1985.[CrossRef][ISI][Medline]
  38. Umekawa T, Yoshida T, Sakane N, Kogure A, Kondo M, and Honjyo H. Trp64Arg mutation of beta3-adrenoceptor gene deteriorates lipolysis induced by beta3-adrenoceptor agonist in human omental adipocytes. Diabetes 48: 117–120, 1999.[Abstract]
  39. Virtanen KA, Rouru J, Hanninen V, Savontaus E, Rouvari T, Teirmaa T, Koulu M, and Huupponen R. Chronic treatment with BRL 35135 potentiates the action of insulin on lipid metabolism. Eur J Pharmacol 332: 215–218, 1997.[CrossRef][ISI][Medline]
  40. West GB, Brown JH, and Enquist BJ. The fourth dimension of life: fractal geometry and allometric scaling of organisms. Science 284: 1677–1679, 1999.[Abstract/Free Full Text]
  41. Weyer C, Tataranni PA, Snitker S, Danforth E Jr, and Ravussin E. Increase in insulin action and fat oxidation after treatment with CL 316,243, a highly selective beta3-adrenoceptor agonist in humans. Diabetes 47: 1555–1561, 1998.[Abstract]
  42. Yoshida T, Sakane N, Wakabayashi Y, Umekawa T, and Kondo M. Anti-obesity effect of CL 316,243, a highly specific beta 3-adrenoceptor agonist, in mice with monosodium-L-glutamate-induced obesity. Eur J Endocrinol 131: 97–102, 1994.[Abstract]
  43. Yoshitomi H, Yamazaki K, Abe S, and Tanaka I. Differential regulation of mouse uncoupling proteins among brown adipose tissue, white adipose tissue, and skeletal muscle in chronic beta 3 adrenergic receptor agonist treatment. Biochem Biophys Res Commun 253: 85–91, 1998.[CrossRef][ISI][Medline]
  44. Young P, Cawthorne MA, and Smith SA. Brown adipose tissue is a major site of glucose utilisation in C57Bl/6 ob/ob mice treated with a thermogenic beta-adrenoceptor agonist. Biochem Biophys Res Commun 130: 241–248, 1985.[CrossRef][ISI][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via ISI Web of Science (4)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kim, H.
Right arrow Articles by LeRoith, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kim, H.
Right arrow Articles by LeRoith, D.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2006 by the American Physiological Society.