The Lack of-Adrenoceptors Results in Enhanced Insulin Sensitivity in ...

Original Article The Lack of ␤-Adrenoceptors Results in Enhanced Insulin Sensitivity in Mice Exhibiting Increased Adiposity and Glucose Intolerance Ce´dric Asensio,1,2 Maria Jimenez,3 Franc¸oise Ku¨hne,1 Franc¸oise Rohner-Jeanrenaud,1,2 and Patrick Muzzin1

We and others have previously shown that triple knockout mice lacking the ␤1/␤2/␤3-adrenoceptors (␤-less mice) developed a progressive obesity at adulthood. Here, we studied the glucose homeostasis in ␤-less mice before the onset of obesity. We show that ␤-less mice have increased fat mass and are glucose intolerant. In addition, we observed that ␤-less mice have impaired glucose-induced insulin secretion and exhibit an increase in liver PEPCK gene expression in the fed state, suggesting that they have increased gluconeogenesis. Although these characteristics are usually associated with insulin resistance, ␤-less mice exhibit enhanced insulin sensitivity during insulin tolerance tests. This is keeping with the results obtained during euglycemic-hyperinsulinemic clamps showing that ␤-less mice display increased insulin responsiveness with normal suppression of hepatic glucose production. Altogether, our results suggest that an intact ␤-adrenergic system is required to regulate overall glucose homeostasis and, in particular, insulin-mediated glucose uptake, most likely at the level of muscles and adipose tissue. Diabetes 54: 3490 –3495, 2005

S

ympathetic activation leads to the local release of catecholamines by the sympathetic nerve terminals and to an increase in circulating catecholamines by the activation of the adrenal medulla. Catecholamines then mediate their effects through ␣- and ␤-adrenoceptor binding on target organs and tissues. The ␣-adrenoceptors are located mainly in the blood vessels and play an important role in cardiovascular regulation. They are also found in adipose tissue and in the pancreas, where they have inhibitory effects on lipolysis and insulin secretion, respectively. In the liver, they mediate the activation of glycogenolysis by catecholamines. The ␤-adrenoceptor family consists of ␤1, ␤2, and ␤3 subtypes. They are expressed in the liver, where they From the 1Department of Cell Physiology and Metabolism, Medical University Centre, Geneva, Switzerland; the 2Department of Internal Medicine, Division of Endocrinology, Diabetology and Nutrition, Medical University Centre, Geneva, Switzerland; and the 3Department of Medical Biochemistry, Medical University Centre, Geneva, Switzerland. Address correspondence and reprint requests to Ce´dric Asensio, Department of Neurology, University of California at San Francisco, Genentech Hall, 600 16th St., 94143-2140, San Francisco, CA. E-mail: [email protected]. Received for publication 7 April 2005 and accepted in revised form 9 September 2005. GTT, glucose tolerance test; ITT, insulin tolerance test. © 2005 by the American Diabetes Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

3490

promote glycogenolysis (1–3), in adipose tissue, where they stimulate lipolysis, and in skeletal muscle. In adipose tissue and skeletal muscle, most studies have reported an inhibition of glucose utilization by the ␤-adrenoceptors (3–7). Most of the knowledge regarding the role of adrenoceptors in glucose homeostasis has been derived from the use of adrenergic agonists and antagonists. One alternative approach to complement the pharmacological tools and further investigate the interactions between the adrenergic system and glucose homeostasis is the suppression of the ␤-adrenoceptors by gene targeting disruption. Recently, the role of ␣1b-adrenoceptor in the control of whole-body glucose homeostasis has been assessed in knockout mice (8). It was found that in the absence of the ␣1b-adrenoceptor, the mice displayed an increased parasympathetic activity leading to hyperinsulinemia and insulin resistance. To further understand the physiological function of the ␤-adrenoceptors in energy metabolism, we generated mice lacking the three ␤-adrenoceptor subtypes, ␤1, ␤2, and ␤3 (␤-less mice). We and others previously reported that these mice become moderately obese with age on chow diet and massively obese on a high-fat diet, despite a normal food intake (9,10). The obesity of ␤-less mice was shown to result from impaired diet-induced thermogenesis (9). ␤-Less mice were also reported to be cold intolerant, failing to initiate cold-induced thermogenesis because of functionally defective brown adipose tissue (9,10). The impact of ablation of ␤-adrenergic signaling on glucose homeostasis has not yet been addressed in this animal model. The aim of the current study was therefore to investigate the role of ␤-adrenoceptors in regulating glucose homeostasis. For this purpose, we assessed glucose tolerance and measured insulin sensitivity and glucose utilization in ␤-less and control mice before the onset of obesity. RESEARCH DESIGN AND METHODS We obtained 3- to 4-month old wild-type and ␤-less mice as previously described (10). Briefly, ␤1⫹/⫺/␤2⫹/⫺/␤3⫹/⫺ mice were crossed to generate ␤1⫹/⫹/␤2⫹/⫹/␤3⫹/⫹ and ␤1⫺/⫺/␤2⫺/⫺/␤3⫺/⫺ mice. Several couples were then established from these homozygous mice, and experiments were performed on ␤1⫹/⫹/␤2⫹/⫹/␤3⫹/⫹ (wild-type) and ␤1⫺/⫺/␤2⫺/⫺/␤3⫺/⫺ (␤-less) offspring. Mice were housed individually and kept on a 12-h light/dark cycle in a temperature-controlled room at 24°C. They were allowed ad libitum access to water and a standard laboratory chow, unless otherwise stated. All procedures used were approved by the Office Ve´te´rinaire Fe´de´ral et Cantonal (Geneva). Dual-energy X-ray absorptiometry. Mice were anesthetized with a solution of ketamine (150 mg/kg body wt) and xylazine (16 mg/kg body wt) and DIABETES, VOL. 54, DECEMBER 2005

C. ASENSIO AND ASSOCIATES

scanned using a densitometer (Lunar Piximus). Fat content was determined as the percentage of body mass. Blood parameters. Blood glucose levels were determined using a glucose meter (One Touch; LifeScan, Milpitas, CA). Plasma insulin concentrations were measured by enzyme-linked immunosorbent assay (Crystal Chem, Chicago, IL). Plasma corticosterone levels were determined by a doubleantibody radioimmunoassay (Immunodiagnostic Systems, Boldon, U.K.). Triglycerides and NEFAs were measured using an enzymatic triglycerides PAP 150 kit (BioMe´rieux, Lyon, France) and a NEFA-C kit (Wako Chemicals, Neuss, Germany), respectively. Hepatic glycogen content. Measurements of glycogen in liver were performed as previously described (11). Total RNA isolation and real-time quantitative PCR. Total RNA was isolated by the method of Chomczynski and Sacchi (12). Oligo-dT first-strand cDNA were synthesized using 5 ␮g total RNA and a Superscript II RNase H reverse transcription kit (Invitrogen Life Technologies, Basel, Switzerland) according to the manufacturer’s instructions. Real-time PCR was performed using an ABI rapid thermal cycler system and a SYBR Green PCR master mix according to the manufacturer’s instructions. The real-time PCR conditions were a step at 50°C for 2 min, followed by a denaturing step at 95°C for 10 min, 40 cycles of denaturation at 95°C for 15 s, and annealing/extension at 60°C for 1 min. A melting curve was generated at the end of the PCR cycles to verify that a single product was amplified. Cyclophilin A was used as a control to account for any variation caused by the efficiencies of the reverse transcription and PCR. The PEPCK oligonucleotide primers used were: upstream 5⬘-tggtggccgtagacctgaa-3⬘ and downstream 5⬘-tgtttgtaggagcagccatgag-3⬘, covering the nucleotides 14391–522 of PEPCK cDNA (GenBank accession no. NM_011044). Cyclophilin A oligonucleotide primers used were: upstream 5⬘-caaatgctggaccaaacacaa-3⬘ and downstream 5⬘-ccatccagccattcagtcttg-3⬘, covering the nucleotides 343– 412 of cyclophilin A (GenBank accession no. XM 355936). The upstream and downstream oligonucleotide primers were chosen on both sides of an intron to prevent amplification of possible contaminating genomic DNA. Glucose tolerance test. After an overnight fast, glucose (1 mg/g body wt) in normal saline (0.9% NaCl) was administered intraperitoneally. Blood glucose levels were measured using a glucose meter (One Touch II; LifeScan) from tail blood at 0, 15, 30, 60, 90, and 120 min after glucose injection. Insulin tolerance test. After a 4-h fast, insulin (0.75 mU/g body wt; Novo Nordisk, Bagsvaerd, Denmark) in normal saline (0.9% NaCl) was administered intraperitoneally. Blood glucose levels were determined using a glucose meter (One Touch II; LifeScan) from the tail blood at 0, 20, 40, 60, and 120 min after insulin injection. Euglycemic-hyperinsulinemic clamps. A catheter was placed into the jugular vein of the mice under anesthesia (pentobarbital, 100 mg/kg i.p.). This catheter was connected to the insulin infusion pump and the glucose and tracer D-[3-3H] glucose infusion pumps (Perkin Elmer Life and Analytical Sciences, Boston, MA). On the day of the experiment, the mice were fasted for 8 h. Mice were continuously infused with the tracer at a rate of 30 ␮Ci 䡠 kg⫺1 䡠 min⫺1 during the whole experiment starting at time ⫽ ⫺45 min. The insulin infusion (18 mU 䡠 kg⫺1 䡠 min⫺1) started at time ⫽ 0 min. Euglycemia was maintained by the use of a concomitant, variable, glucose (20% solution) infusion. Glycemia was determined in 3.5 ␮l of blood sampled from the tip of the tail vein every 5 min using a glucose meter (One Touch II; LifeScan). In addition, 5 ␮l of blood was recovered at time ⫽ ⫺10, 0, 50, and 60 min, and specific activity of the tracer was measured in deproteinized blood samples, as previously reported (13). Whole-body glucose turnover was then calculated by dividing the tracer infusion rate by the plasma glucose specific activity. Hepatic glucose production was calculated as the difference between the glucose turnover rate (Rd) and the glucose infusion rate in steady-state conditions. Glucose-induced insulin secretion. After an overnight fast, glucose (1 mg/g body wt) in normal saline (0.9% NaCl) was administered intraperitoneally. Blood glucose and plasma insulin levels were measured from the tail blood at 0, 5, 15, 30, 60, and 120 min after glucose injection. Statistical methods. The results are given as the means ⫾ SE. Statistical analysis was performed using the Mann-Whitney rank sum test or the two-tailed unpaired Student’s t test, except for food restriction experiments, which were analyzed using paired Student’s t test. The same group of wild-type or ␤-less mice was used for the insulin tolerance test (ITT) in ad libitum and restricted states. The calculations were performed using Sigma STAT software (SPSS, Chicago, IL). A P value ⬍0.05 was considered statistically significant.

TABLE 1 Body weight, food intake, and fat mass

Body weight (g) Food intake (g/day) Fat mass (%) WATe (mg)

Wild type

␤-Less

30.2 ⫾ 0.6 4.0 ⫾ 0.1 19.8 ⫾ 1.2 573 ⫾ 72

30.0 ⫾ 0.5 3.8 ⫾ 0.1 30.6 ⫾ 0.5* 806 ⫾ 70*

Data are means ⫾ SE. n ⫽ 10 per group, except for the determination of epididymal fat pad mass, where n ⫽ 6. *P ⬍ 0.05 vs. wild type. WATe, epididymal fat pad.

wild-type mice. At this age, there was no difference in body weight, nor in food intake between the two groups of mice (Table 1). However, dual-energy X-ray absorptiometry analysis revealed that the percentage of body fat of ␤-less mice was highly increased in comparison with wild-type mice. Corroborating this result, the weight of the epididymal fat pad was found to be increased in ␤-less mice (Table 1). ␤-Less mice are glucose intolerant and display impaired glucose-induced insulin secretion. We then looked at whether glucose tolerance was altered in ␤-less mice. For this purpose, we performed glucose tolerance tests (GTTs) in the two groups of mice. As shown in Fig. 1, the glycemia of ␤-less mice was significantly higher in comparison with controls during the GTT. We reasoned that the glucose intolerance of ␤-less mice could be explained either by a defect in glucose-induced insulin secretion and/or by a decrease in insulin sensitivity. To test the first hypothesis, we looked at potential changes in insulin levels after an intraperitoneal bolus of glucose (1 mg/g). In the fasted state, we observed that the insulin levels of ␤-less mice were not statistically different from those of wild-type mice (0.40 ⫾ 0.07 and 0.56 ⫾ 0.04 ng/ml in wild-type and ␤-less mice, respectively; NS). In contrast, the increase over basal insulin levels was significantly lower in ␤-less than in control mice after the glucose bolus, and the difference was maintained at 120 min postinjection (Fig. 2). ␤-Less mice have increased hepatic PEPCK mRNA expression and are hypercorticosteronemic. Because the physiological situation corresponding to the glucose administration is the fed state and because ␤-less mice showed impaired insulin release in response to a glucose

RESULTS

Body composition of ␤-less mice is altered. We studied 3- to 4-month-old triple knockout male mice lacking the ␤1/␤2/␤3-adrenoceptors (␤-less) and corresponding control DIABETES, VOL. 54, DECEMBER 2005

FIG. 1. GTT. Blood glucose levels during intraperitoneal GTT (1 mg/g body wt) in wild-type and ␤-less mice are shown. Values are the means ⴞ SE, n ⴝ 7 animals per group. *P < 0.05. F, wild-type mice; E, ␤-less mice. 3491

GLUCOSE HOMEOSTASIS IN ␤-LESS MICE

FIG. 2. Glucose-induced insulin secretion. Changes in plasma insulin levels over basal levels after an intraperitoneal bolus of glucose (1 mg/g body wt) in wild-type and ␤-less mice are shown. Inset: Areas under the curve of the mean values. Values are the means ⴞ SE, n ⴝ 5– 8 animals per group. *P < 0.05. F, wild-type mice; E, ␤-less mice.

challenge, we examined whether the lack of insulin secretion during the fed state would lead to an increase in hepatic glucose production. For this purpose, we examined the mRNA expression of PEPCK, a key gluconeogenic enzyme whose expression has been shown to be suppressed by insulin (14) in wild-type and ␤-less fed mice. Using quantitative real-time PCR, we observed that PEPCK mRNA levels were 1.5-fold higher in ␤-less than in wild-type mice (Fig. 3A). No difference in hepatic glycogen content was found between the two groups of mice (37.8 ⫾ 4.3 and 31.8 ⫾ 7.6 mg/g of liver in wild-type and ␤-less mice, respectively; NS), suggesting that hepatic glycogenolysis was not different between ␤-less and wildtype mice. Because glucocorticoids are also important regulators of hepatic glucose output via their stimulatory effects on gluconeogenesis, we measured corticosterone plasma levels in fed wild-type and ␤-less mice. We found that ␤-less mice have increased glucocorticoid levels (Fig. 3B). In contrast, free fatty acid (0.53 ⫾ 0.02 and 0.53 ⫾ 0.07 mEq/l in wild-type and ␤-less mice, respectively; NS) and triglyceride (128.3 ⫾ 16.2 and 101.2 ⫾ 10.9 mg/dl, respectively; NS) concentrations were not significantly different between wild-type and ␤-less mice. ␤-Less mice have increased insulin sensitivity. To test the second hypothesis that may also explain the glucose intolerance, namely a decrease in insulin sensitivity in ␤-less mice, we performed ITTs in the two groups of mice. Surprisingly, as shown in Fig. 4, the fall of glycemia after insulin administration was of greater magnitude in ␤-less than in wild-type mice. Moreover, the area under the ITT curve of ␤-less mice was significantly smaller than that of wild-type mice (Fig. 4, inset). Dietary restriction has been shown to increase insulin sensitivity. Because ␤-less mice displayed increased insulin sensitivity, we hypothesized that the beneficial effect of food restriction on whole-body insulin-mediated glucose clearance, which is thought to be mediated, at least in part, by a decrease in sympathetic tone, would be less pronounced in ␤-less than in wild-type mice. As shown in Fig. 5A, we observed that food restriction (20% reduction for 6 days) was able to ameliorate insulin sensitivity of wild3492

FIG. 3. PEPCK gene expression in liver and plasma corticosterone levels. A: PEPCK mRNA levels in wild-type and ␤-less mice. The expression of PEPCK relative to that of cyclophilin was determined by real-time PCR analysis. The ratio of the wild-type values is considered to be 1.0. B: Plasma corticosterone in wild-type and ␤-less mice. All values are the means ⴞ SE, n ⴝ 6 animals per group. *P < 0.05. f, wild-type mice; 䡺, ␤-less mice.

type animals, but it had no effect on this parameter in ␤-less mice (Fig. 5B). To further substantiate the observations made with the ITTs, we performed euglycemic-hyperinsulinemic clamps. The metabolic parameters before and at the end of the clamps are shown in Table 2. During the clamps, we observed that a higher glucose infusion rate was required

FIG. 4. ITT. Blood glucose levels expressed as percent basal glycemia during intraperitoneal ITTs (0.75 mU/g body wt) in wild-type and ␤-less mice. Values are the means ⴞ SE, n ⴝ 6 –7 animals per group. Inset: Areas under the curve of the mean values expressed as percent basal glycemia. *P < 0.05. F, wild-type mice; E, ␤-less mice. DIABETES, VOL. 54, DECEMBER 2005

C. ASENSIO AND ASSOCIATES

FIG. 5. Effect of food restriction on ITT in wild-type and ␤-less mice. A: Blood glucose levels expressed as percent basal glycemia during intraperitoneal ITT (0.75 mU/g body wt) before (F) and after (U) food restriction in wild-type mice. Values are the means ⴞ SE, n ⴝ 6 –7 animals per group. Inset: Areas under the curve of the mean values expressed as percent basal glycemia of fed and food-restricted wildtype mice. *P < 0.05. B: Blood glucose levels expressed as percent basal glycemia during intraperitoneal ITT before (E) and after (ƒ) food restriction in ␤-less mice. Values are the means ⴞ SE, n ⴝ 6 animals per group. Inset: Areas under the curve of the mean values expressed as percent basal glycemia of fed and food-restricted ␤-less mice.

to maintain normal glucose levels in ␤-less compared with control mice (23.2 ⫾ 1.4 and 32.2 ⫾ 3.0 mg 䡠 min⫺1 䡠 kg⫺1 in wild-type and ␤-less mice, respectively; P ⬍ 0.05). As shown in Fig. 6A, this difference was caused by an increase in the rate of glucose disappearance (Rd), whereas hepatic glucose production was similarly suppressed in both groups (Fig. 6B). DISCUSSION

We have shown that 3- to 4-month-old mice lacking the ␤1/␤2/␤3-adrenoceptors are glucose intolerant compared with wild-type controls, as demonstrated by the results of

FIG. 6. Euglycemic-hyperinsulinemic clamps. A: Rate of glucose disappearance (Rd) at the end of euglycemic-hyperinsulinemic clamps in wild-type and ␤-less mice. Values are means ⴞ SE, n ⴝ 5– 6 animals per group. *P < 0.05. B: Hepatic glucose production (HGP) at the end of euglycemic-hyperinsulinemic clamps in wild-type and ␤-less mice. Values are the means ⴞ SE, n ⴝ 5– 6 animals per group. f, wild-type mice; 䡺, ␤-less mice.

the GTT. We reasoned that this observation could be caused either by a defect in glucose-induced insulin secretion and/or by a decrease in insulin sensitivity. Concerning the former hypothesis, we observed that ␤-less mice actually display a defect in insulin secretion. This last observation is consistent with previous results from our laboratory showing that high fat–fed mice lacking the ␤3-adrenoceptor also exhibit a glucose intolerance caused by impaired insulin secretion (M.J., unpublished data). In humans, the ␤3-adrenoceptor has been shown to be expressed in the pancreatic islets, and it has been proposed that its activation stimulates insulin secretion by ␤-cells (15). Thus, it is possible that the lack of ␤3adrenoceptors is directly responsible for the impaired insulin secretion observed in our mice. However, it should be stated that ␤-cell–specific ␤3-adrenergic receptor ex-

TABLE 2 Various metabolic parameters before and during euglycemic-hyperinsulinemic clamps Glucose (mg/dl) Wild type ␤-Less

84.9 ⫾ 12.6 60.6 ⫾ 8.4

Basal period Insulin (ng/ml) 1.4 ⫾ 0.1 1.4 ⫾ 0.2

Clamp period HGP (mg 䡠 min⫺1 䡠 kg⫺1)

Glucose (mg/dl)

Insulin (ng/ml)

11.0 ⫾ 1.1 9.8 ⫾ 0.9

74.9 ⫾ 7.7 74.8 ⫾ 8.0

10.1 ⫾ 1.0 10.6 ⫾ 1.3

Data are the means ⫾ SE. n ⫽ 6 for wild type, n ⫽ 5 for ␤-less. No intergroup difference. HGP, hepatic glucose production. DIABETES, VOL. 54, DECEMBER 2005

3493

GLUCOSE HOMEOSTASIS IN ␤-LESS MICE

pression has not yet been clearly demonstrated in mice (M.J., unpublished data) (16). Because ␤1/␤2-adrenergic stimulation of ␤-cells is thought not to play a major role in controlling insulin secretion, we can speculate that the observed effect results from the activation of the ␤-adrenergic system on tissues that may indirectly regulate insulin secretion. With regard to this possibility, it has been proposed that a factor secreted by white adipose tissue, whose expression is under the regulation of the ␤3adrenoceptor, could mediate the ␤-adrenergic effect on insulin secretion (16). Finally, we could also suppose that there is an upregulation of the ␣2-adrenoceptors in the pancreatic ␤-cells of our ␤-less mice. Because these receptors are known to inhibit insulin secretion, they could be responsible for the observed defect. In addition, together with the defect of glucose-induced insulin secretion, we observed that, in a fed state, PEPCK mRNA levels were 1.5-fold higher in the liver of ␤-less mice than in controls. This increase suggests that the lack of glucose-induced insulin secretion in ␤-less mice during the GTT leads to a less efficient inhibition of PEPCK gene expression in the liver. Another possible explanation for the increase of PEPCK expression in ␤-less mice is the hypercorticosteronemia that we observed in these animals. Indeed, glucocorticoids are known to stimulate hepatic expression of the PEPCK gene both via a glucocorticoid responsive element on its promoter and through the recruitment of proliferator-activated receptor-␥ coactivator (17,18). Concerning a possible involvement of a defect in insulin sensitivity for explaining the glucose intolerance of ␤-less mice, we observed that ␤-less mice are surprisingly more sensitive to insulin, as demonstrated by the results of the euglycemic-hyperinsulinemic clamps and ITTs performed in fed wild-type and ␤-less mice, as well as by those obtained in food-restricted mice. Indeed, in the present study, food restriction, which is thought to decrease sympathetic activity in both humans and rodents (19 –21), ameliorated insulin tolerance of wild-type mice, whereas it had no effect on insulin sensitivity in ␤-less animals. Taken together, these results suggested that ␤-adrenoceptors are essential in the regulation of insulin-mediated glucose utilization. The enhanced insulin sensitivity of ␤-less mice is surprising because these animals display increased fat mass, which has been shown to be one of the major factors contributing to the development of insulin resistance. However, it has also been reported that ␤-adrenergic stimulation inhibits insulin-stimulated glucose uptake in skeletal muscle and in white adipose tissue. Indeed, it has been shown that epinephrine decreases insulin-stimulated glucose transport in rat skeletal muscle and in humans by altering the intrinsic activity of surface GLUT4 transporters (22–25). In addition, the ␤-adrenergic agonist isoproterenol was shown to inhibit insulin-stimulated glucose transport in rat adipose cells (4,5). Thus, it is possible that the suppression of ␤-adrenergic stimulation directly contributes to the enhanced peripheral insulin sensitivity of ␤-less mice. The augmentation in insulin sensitivity is of particular interest with regard to the use of ␤-blockers in humans. It has been shown that they are highly effective in reducing the risk of cardiovascular events and death in post– myocardial infarction patients with diabetes (26). Indeed, the insulin resistance associated with type 2 diabetes leads to high insulin levels, resulting in increased sympathetic activation and thus norepinephrine release (27–30). Fur3494

thermore, norepinephrine has been shown to cause hypertension, to increase the risk of sudden death by lowering the threshold to lethal ventricular arrhythmias, and to augment arterial wall damage predisposing to atheroma. In this context, ␤-blockade could inhibit the harmful actions of sustained high norepinephrine activity (31). It would therefore be interesting to further explore whether our ␤-less mice have enhanced cardiovascular protection. However, this potential benefit of the ␤-blockade could be to the detriment of obesity and glucose intolerance, as suggested by the phenotype of ␤-less mice. Altogether, our results obtained with ␤-less mice show that an intact ␤-adrenergic system is required to regulate overall glucose homeostasis, and they complement previous data obtained with pharmacological agonists or antagonists. In particular, the glucose intolerance of ␤-less mice seems to be related to defective glucose-induced insulin secretion as well as a glucocorticoid-induced increase in gluconeogenesis. In addition, ␤-less mice are more sensitive to insulin in terms of the rate of glucose utilization (Rd). Future studies in insulin-sensitive tissues of ␤-less mice could provide important information on the cross talk between the ␤-adrenergic and insulin signaling pathways. ACKNOWLEDGMENTS

This work was supported by Swiss National Science Foundation Grant 3100AO-105889 (to F.R.-J.), the Fondation Boninchi (to P.M.), the Fondation du Centenaire de la Socie´te´ Suisse d’Assurances Ge´ne´rales sur la Vie Humaine Pour la Sante´ Publique et les Recherches Me´dicales (to P.M.), and European Community FP6 contract LSHM-CT2003-503041 (to F.R.-J.). This study was part of the Geneva Programme for Metabolic Disorders. We are indebted to Pr. Jean-Paul Giacobino for helpful discussions and critical comments of this manuscript, and we thank Dr. Dominique Pierroz for giving us access to the dual-energy X-ray absorptiometry scanner. We are grateful to Dr. Kobilka for providing us with the double-knockout mice. Finally, we thank Marcella Klein for her excellent technical assistance. REFERENCES 1. Arinze IJ, Kawai Y: Adrenergic regulation of glycogenolysis in isolated guinea-pig hepatocytes: evidence that beta 2-receptors mediate catecholamine stimulation of glycogenolysis. Arch Biochem Biophys 225:196 –202, 1983 2. Lum BK, Lau YS, Buesa R, Lockwood RH, Kuo SH: Studies on the hyperglycemia and hepatic glycogenolysis produced by alpha and beta adrenergic agonists in the cat. Life Sci 26:1195–1202, 1980 3. Rizza RA, Cryer PE, Haymond MW, Gerich JE: Adrenergic mechanisms for the effects of epinephrine on glucose production and clearance in man. J Clin Invest 65:682– 689, 1980 4. Nishimura H, Saltis J, Habberfield AD, Garty NB, Greenberg AS, Cushman SW, Londos C, Simpson IA: Phosphorylation state of the GLUT4 isoform of the glucose transporter in subfractions of the rat adipose cell: effects of insulin, adenosine, and isoproterenol. Proc Natl Acad Sci U S A 88:11500 – 11504, 1991 5. Yang J, Hodel A, Holman GD: Insulin and isoproterenol have opposing roles in the maintenance of cytosol pH and optimal fusion of GLUT4 vesicles with the plasma membrane. J Biol Chem 277:6559 – 6566, 2002 6. Sloan IG, Sawh PC, Bihler I: Influence of adrenalin on sugar transport in soleus, a red skeletal muscle. Mol Cell Endocrinol 10:3–12, 1978 7. Abramson EA, Arky RA: Role of ␤-adrenergic receptors in counterregulation to insulin-induced hypoglycemia. Diabetes 17:141–146, 1968 8. Burcelin R, Uldry M, Foretz M, Perrin C, Dacosta A, Nenniger-Tosato M, Seydoux J, Cotecchia S, Thorens B: Impaired glucose homeostasis in mice lacking the alpha1b-adrenergic receptor subtype. J Biol Chem 279:1108 – 1115, 2004 9. Bachman ES, Dhillon H, Zhang CY, Cinti S, Bianco AC, Kobilka BK, Lowell DIABETES, VOL. 54, DECEMBER 2005

C. ASENSIO AND ASSOCIATES

BB: betaAR signaling required for diet-induced thermogenesis and obesity resistance. Science 297:843– 845, 2002 10. Jimenez M, Leger B, Canola K, Lehr L, Arboit P, Seydoux J, Russell AP, Giacobino JP, Muzzin P, Preitner F: Beta(1)/beta(2)/beta(3)-adrenoceptor knockout mice are obese and cold-sensitive but have normal lipolytic responses to fasting. FEBS Lett 530:37– 40, 2002 11. Chan TM, Exton JH: A rapid method for the determination of glycogen content and radioactivity in small quantities of tissue or isolated hepatocytes. Anal Biochem 71:96 –105, 1976 12. Chomczynski P, Sacchi N: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156 –159, 1987 13. Vettor R, Zarjevski N, Cusin I, Rohner-Jeanrenaud F, Jeanrenaud B: Induction and reversibility of an obesity syndrome by intracerebroventricular neuropeptide Y administration to normal rats. Diabetologia 37:1202– 1208, 1994 14. Barthel A, Schmoll D: Novel concepts in insulin regulation of hepatic gluconeogenesis. Am J Physiol Endocrinol Metab 285:E685–E692, 2003 15. Perfetti R, Hui H, Chamie K, Binder S, Seibert M, McLenithan J, Silver K, Walston JD: Pancreatic beta-cells expressing the Arg64 variant of the beta(3)-adrenergic receptor exhibit abnormal insulin secretory activity. J Mol Endocrinol 27:133–144, 2001 16. Grujic D, Susulic VS, Harper ME, Himms-Hagen J, Cunningham BA, Corkey BE, 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 17. Hanson RW, Reshef L: Regulation of phosphoenolpyruvate carboxykinase (GTP) gene expression. Annu Rev Biochem 66:581– 611, 1997 18. Yoon JC, Puigserver P, Chen G, Donovan J, Wu Z, Rhee J, Adelmant G, Stafford J, Kahn CR, Granner DK, Newgard CB, Spiegelman BM: Control of hepatic gluconeogenesis through the transcriptional coactivator PGC-1. Nature 413:131–138, 2001 19. Young JB, Landsberg L: Suppression of sympathetic nervous system during fasting. Science 196:1473–1475, 1977 20. Leibel RL, Berry EM, Hirsch J: Metabolic and hemodynamic responses to

DIABETES, VOL. 54, DECEMBER 2005

endogenous and exogenous catecholamines in formerly obese subjects. Am J Physiol 260:R785–R791, 1991 21. Arrone LJ, Mackintosh R, Rosenbaum M, Leibel RL, Hirsch J: Cardiac autonomic nervous system activity in obese and never-obese young men. Obes Res 5:354 –359, 1997 22. Han XX, Bonen A: Epinephrine translocates GLUT-4 but inhibits insulinstimulated glucose transport in rat muscle. Am J Physiol 274:E700 –E707, 1998 23. Bonen A, Megeney LA, McCarthy SC, McDermott JC, Tan MH: Epinephrine administration stimulates GLUT4 translocation but reduces glucose transport in muscle. Biochem Biophys Res Commun 187:685– 691, 1992 24. Jones JP, Dohm GL: Regulation of glucose transporter GLUT-4 and hexokinase II gene transcription by insulin and epinephrine. Am J Physiol 273:E682–E687, 1997 25. Laurent D, Petersen KF, Russell RR, Cline GW, Shulman GI: Effect of epinephrine on muscle glycogenolysis and insulin-stimulated muscle glycogen synthesis in humans. Am J Physiol 274:E130 –E138, 1998 26. Kjekshus J, Gilpin E, Cali G, Blackey AR, Henning H, Ross J Jr: Diabetic patients and beta-blockers after acute myocardial infarction. Eur Heart J 11:43–50, 1990 27. Tack CJ, Smits P, Willemsen JJ, Lenders JW, Thien T, Lutterman JA: Effects of insulin on vascular tone and sympathetic nervous system in NIDDM. Diabetes 45:15–22, 1996 28. Lembo G, Napoli R, Capaldo B, Rendina V, Iaccarino G, Volpe M, Trimarco B, Sacca L: Abnormal sympathetic overactivity evoked by insulin in the skeletal muscle of patients with essential hypertension. J Clin Invest 90:24 –29, 1992 29. Paolisso G, Manzella D, Tagliamonte MR, Rizzo MR, Gambardella A, Varricchio M: Effects of different insulin infusion rates on heart rate variability in lean and obese subjects. Metabolism 48:755–762, 1999 30. Arauz-Pacheco C, Lender D, Snell PG, Huet B, Ramirez LC, Breen L, Mora P, Raskin P: Relationship between insulin sensitivity, hyperinsulinemia, and insulin-mediated sympathetic activation in normotensive and hypertensive subjects. Am J Hypertens 9:1172–1178, 1996 31. Cruickshank JM: Beta-blockers and diabetes: the bad guys come good. Cardiovasc Drugs Ther 16:457– 470, 2002

3495