Chronic antidiabetic and cardiovascular actions of leptin: role of CNS and increased adrenergic activity

Transcription

1 Am J Physiol Regul Integr Comp Physiol 291: R1275 R1282, First published June 15, 2006; doi: /ajpregu Chronic antidiabetic and cardiovascular actions of leptin: role of CNS and increased adrenergic activity Alexandre A. da Silva, Lakshmi S. Tallam, Jiankang Liu, and John E. Hall Department of Physiology and Biophysics, Center of Excellence in Cardiovascular-Renal Research, University of Mississippi Medical Center, Jackson, Mississippi Submitted 15 May 2006; accepted in final form 13 June 2006 da Silva, Alexandre A., Lakshmi S. Tallam, Jiankang Liu, and John E. Hall. Chronic antidiabetic and cardiovascular actions of leptin: role of CNS and increased adrenergic activity. Am J Physiol Regul Integr Comp Physiol 291: R1275 R1282, First published June 15, 2006; doi: /ajpregu This study examined the importance of direct central nervous system (CNS) actions and increased adrenergic activity in mediating the chronic antidiabetic and cardiovascular actions of leptin. Insulin-deficient rats (streptozotocin, 50 mg/kg) were used to examine the effects of leptin on glucose homeostasis independent of changes in insulin. Male Sprague-Dawley rats were instrumented with arterial and venous catheters and intracerebroventricular cannula for 24-h/day blood pressure (BP) and heart rate (HR) monitoring and intravenous and intracerebroventricular infusions. Insulin-deficient diabetes was associated with marked hyperglycemia, hyperphagia, decreased BP, and pronounced fall in HR. Leptin treatment, intravenous or intracerebroventricular, completely restored to control values plasma glucose levels ( to and to 65 7 mg/dl, respectively), food intake, BP, and HR (304 8to364 7and to bpm, respectively). Combined blockade of 1 -, 1 -, and 2 -adrenergic receptors attenuated the rise in HR by 30 to 50% but had no effect on the antidiabetic and dietary actions of leptin. Blockade of 3 -adrenergic receptors did not attenuate the chronic cardiovascular or metabolic effects of leptin. These data demonstrate that leptin, via its direct actions in the CNS, has powerful antidiabetic actions in insulin-deficient rats independent of increased peripheral 1, 1, 2, and 3 -adrenergic activity. Leptin also exerts important long-term cardiovascular actions that are partially mediated via 1 - and 1 / 2 -adrenergic activation. These findings provide new insights into novel pathways for long-term control of glucose homeostasis and cardiovascular regulation. glucose; insulin; blood pressure; food intake; diabetes; sympathetic activity; central nervous system; heart rate; kidney; appetite LEPTIN, AN ADIPOCYTE-DERIVED peptide, regulates body weight and body fat mass by reducing appetite and increasing energy expenditure (10, 11, 28). Emerging evidence, however, indicates that leptin also has important antidiabetic and cardiovascular effects. For example, leptin administration enhances insulin-stimulated glucose uptake in peripheral tissues and decreases plasma insulin levels in normal rats (2, 4, 29, 34) and improves insulin sensitivity in humans and mice with lipodystrophy characterized by severe insulin resistance (24, 30). Improved insulin sensitivity enhances insulin-mediated suppression of glucose output by the liver and increases glucose uptake by tissues such as skeletal muscle and fat. The overall effect is to reduce plasma glucose and/or the plasma level of insulin required to maintain euglycemia. Leptin may also have important effects on glucose homeostasis that are independent of insulin. For example, leptin injections in insulin-deficient diabetic animals restored plasma glucose levels to normal levels even though plasma insulin levels were nearly undetectable (5). Although the powerful effects of leptin on glucose regulation have been clearly demonstrated, the mechanisms involved have not been elucidated. In addition to its metabolic effects, leptin may also be important in cardiovascular regulation via its effects on the central nervous system (CNS). Acute intracerebroventricular leptin infusion increases sympathetic nervous system (SNS) activity in various tissues, and chronic hyperleptinemia increases heart rate (HR) and arterial blood pressure (BP; 8, 29). The chronic effects of leptin to raise BP are abolished by and -adrenergic blockade, indicating that they are mediated by adrenergic activation (4). Current evidence suggests that leptin s effects on SNS activity may be mediated, at least in part, by activation of the same hypothalamic neurons [e.g., proopiomelanocortin (POMC) neurons] that regulate appetite (6, 27). Activation of hypothalamic neurons and increased SNS activity may provide a common CNS pathway for leptin to exert its antidiabetic and cardiovascular actions. However, the importance of the direct CNS actions of leptin in long-term control of glucose and cardiovascular regulation is unclear. Also, the potential mechanisms by which the CNS communicates with peripheral tissues to influence glucose homeostasis have not been elucidated, although SNS and adrenergic activation have been suggested to play a key role (1, 3, 19, 20). The aim of this study was to first determine the importance of CNS mechanisms in mediating the chronic actions of leptin on regulation of glucose, BP, and HR. Insulin-deficient diabetic rats were used to examine the effects of leptin on glucose homeostasis independent of changes in insulin. We then assessed the role of 1 -, 1 -, 2 -, and 3 -adrenergic receptors in mediating the CNS effects of leptin to alter glucose and cardiovascular regulation. Our results indicate that most of leptin s chronic antidiabetic actions can be explained largely by direct effects on the hypothalamus. Although - and adrenergic activation mediate much of the chronic CNS effects of leptin on BP and HR, increased adrenergic activity does not appear to be the most important pathway by which the CNS influences peripheral glucose homeostasis. Address for reprint requests and other correspondence: A. A. da Silva, Dept. of Physiology and Biophysics, Univ. of Mississippi Medical Center, 2500 North State St., Jackson, MS ( asilva@physiology. umsmed.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 /06 $8.00 Copyright 2006 the American Physiological Society R1275

2 R1276 METHODS Animals Male Sprague-Dawley rats (n 33, Harlan, Indianapolis, IN) weighing between 275 to 325 g were used in these studies. The experimental procedures and protocols of this study conform to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the University of Mississippi Medical Center. Surgical Procedures Rats were anesthetized with 50 mg/kg pentobarbital sodium (Nembutal), and atropine sulfate (0.1 mg/kg) was administered to prevent excess airway secretions. Arterial and venous catheters were implanted according to procedures previously described (4, 29). Briefly, using aseptic techniques, a laparotomy was performed, and a sterile nonocclusive polyvinyl catheter was inserted into the abdominal aorta, distal to the kidneys. Through a left femoral vein incision, a sterile catheter was placed in the vena cava. Both catheters were exteriorized through a subcutaneously implanted stainless steel button. Immediately after arterial and venous catheter implantation, a stainless steel cannula (21 gauge, 10 mm long) was implanted into the right lateral cerebral ventricle using the coordinates as previously described (6, 7, 16). Accuracy of cannula placement was tested by measuring the dipsogenic response (immediate drinking of at least 5 ml of water in 10 min) to an intracerebroventricular injection of ANG II (10 nmol in 5 l). Water and Electrolyte Balances and Hemodynamic Measurements After recovery from anesthesia, rats were housed individually in metabolic cages for determination of daily water and electrolyte intake and urine output. The arterial and venous catheters were connected to a dual-channel infusion swivel (Instech). The arterial catheter was connected to a pressure transducer (Maxxim) for continuous 24-h measurement of MAP and HR using computerized techniques, as previously described (29). The venous catheter was connected to a syringe pump for continuous infusion of saline (0.45%, 40 ml/day). The rats received food and water ad libitum throughout the study. Total sodium intake was maintained constant at 3.1 meq/day via the continuous saline infusion combined with sodiumdeficient rat chow (0.006 mmol sodium/g food, Harlan Teklad). Intravenous solutions were infused through a sterile filter (0.22 m, Millipore), and the saline infusion was started immediately after placement of the rats into the metabolic cages. The rats were allowed to recover for 7 10 days before control measurements were recorded. Experimental Protocols MAP, HR, urine volume, food, and water intakes were recorded daily, and urine samples were collected for analysis of electrolytes. Blood samples (1.5 ml) were collected on the 4th day of control period, on the 4th day after streptozotocin (STZ) injection, on the 10th day of leptin treatment and on the 4th day of the recovery period for measurements of glomerular filtration rate (GFR) and plasma insulin, glucose, and leptin concentrations. The blood was replaced with 1.5 ml of saline 0.9%. Induction of diabetes. After 4 to 5 days of stable control measurements, insulin-deficient diabetes was induced by a single intravenous injection of STZ (50 mg/kg, Sigma-Aldrich, dissolved in 0.5 ml of 0.05 M citrate buffer, ph 4.5). Chronic intracerebroventricular or intravenous leptin infusion in diabetic rats. Five days after STZ injection, rats received a continuous 24-h/day infusion of either 1) leptin (1 g kg 1 min 1 iv, R&D Systems, n 5) added to the intravenous saline infusion, or 2) leptin (0.021 g kg 1 min 1 icv, n 5) via osmotic minipump (0.5 l/h, Durect) implanted in the scapular region, for a period of 10 days followed by a 5-day recovery period. Diabetic rats receiving only saline intravenous infusion were used as controls (n 5). Chronic intracerebroventricular leptin infusion during adrenergic receptor blockade. Five days after STZ injection, another group of rats received either 1) a mixture of an 1 adrenergic receptor antagonist, terazosin (10 mg kg 1 day 1, Sigma), plus a 1/ 2 adrenergic receptor antagonist, propranolol (10 mg kg 1 day 1, Sigma; n 5); or 2) a selective 3-adrenergic receptor antagonist, SR59230A (5 mg kg 1 day 1, Sigma; n 5), for 20 days. The compounds were added to the intravenous saline infusion for continuous infusion 24 h/day. Five days after the infusion of antagonist was started, rats received a intracerebroventricular leptin infusion at g kg 1 min 1 via osmotic minipumps for 10 days. The doses of the antagonists were chosen on the basis of previous studies showing blockade of at least 90 95% of the responses to bolus injection of adrenergic receptors agonists (4, 25). Chronic 3-adrenergic receptor activation. To further investigate the role of 3-adrenergic receptor activation in mediating the antidiabetic actions of leptin mediated by the CNS, we studied an additional group of insulin-deficient diabetic rats that was infused with a selective 3-receptor agonist, CL316,243 (1 mg kg 1 day 1 iv, Sigma, n 4), for 10 days starting at day 5 after STZ injection. This dose has been shown to reduce glucose and insulin levels in obese Zucker rats (19). Analytical Methods Plasma insulin and leptin concentrations were measured by radioimmunoassay. Plasma glucose concentration was measured using the glucose oxidation method (Beckman glucose analyzer 2), and small blood samples were taken each morning between 9:00 and 10:00 AM for determination of blood glucose levels using glucose strips. GFR was calculated from the 24-h clearance of [ 125 I]-labeled iothalamate, as previously described (29). Statistical Analysis The data are expressed as means SE and analyzed by using two-factor ANOVA with repeated measures. The Bonferroni post hoc test was used for comparisons between groups. Dunnett s test was used for comparisons of experimental and control values within each group when the overall preceding ANOVA analyses demonstrated statistical significance. Statistical significance was accepted at a level of P RESULTS Metabolic, Hormonal, and Renal Changes in Diabetic Rats During Chronic Intracerebroventricular or Intravenous Leptin Infusion A single STZ injection decreased plasma insulin to very low levels (Table 1) with consequent development of hyperglycemia and increases in food intake (Fig. 1A). Intravenous leptin infusion for 10 days lowered food intake to control values (Fig. 1A) and reduced blood glucose to nondiabetic levels (from to mg/dl, Fig. 1B). Chronic intracerebroventricular leptin infusion directly into the CNS at a rate of g kg 1 min 1, only about 2% of the intravenous infusion rate (1.0 g kg 1 min 1 ), reduced plasma glucose from to 65 7 mg/dl (Fig. 1C). Chronic intracerebroventricular leptin infusion resulted in only a very small increases in plasma leptin levels compared with control levels before infusion ( to ng/ml), whereas intravenous leptin infusion caused a large increase in circulating leptin levels (from to ng/ml, Table 1). These data indicate that there was no major spillover into

3 Table 1. Effects of intravenous (1.0 g kg 1 min 1 ) and intracerebroventricular (0.021 g kg 1 min 1 ) leptin or vehicle infusion on plasma hormones, renal function, and water intake in streptozotocin-induced diabetic rats R1277 Experimental Groups Plasma Insulin, U/ml Plasma Leptin, ng/ml GFR, ml/min Urine Volume, ml/day Water Intake, ml/day Saline vehicle Control STZ - day 4 3 1* * * * * STZ - day * * * * * IV leptin Control STZ - day 4 3 1* * * * STZ leptin - day * * * 26 7* STZ - recovery 7 4* * * * * ICV leptin Control STZ - day 4 4 2* * * * * STZ leptin - day * * * 20 4* STZ - recovery 6 2* * * 66 18* 36 12* Values are presented as means SE. Values are given for day 4 of control, day 4 after STZ injection, experimental day 10, and day 4 of recovery period. *P 0.05 compared with control. GFR, glornerular filtration rate. the systemic circulation of leptin administered intracerebroventricularly and that the effects of ICV leptin were mediated via CNS pathways. Plasma insulin levels were not altered and remained very low during intracerebroventricular or intravenous leptin infusions (Table 1). GFR increased (P 0.05) during the onset of diabetes (Table 1), and leptin infusion prevented the increase in GFR (Table 1). Daily urine output and water intake also increased (P 0.001) after STZ injection (Table 1) and returned to control values with chronic intracerebroventricular or intrave- Fig. 1. Responses to chronic intravenous leptin (1.0 g kg 1 min 1 ) or vehicle infusion on food intake (A) and on blood glucose levels (B), and chronic intracerebroventricular leptin infusion (0.021 m kg 1 min 1 ) or vehicle infusion in pair-fed rats on blood glucose levels (C) and on food intake (D) in streptozotocin-treated Sprague-Dawley rats. Data represent means SE of 5 rats in each group. Two-factor ANOVA for repeated measures with Bonferroni test for comparisons between groups and Dunnett s test for comparisons of experimental and control values within each group were used.

4 R1278 nous leptin treatment (Table 1), which may be attributed to the normalization of plasma glucose levels. After cessation of leptin infusion, plasma glucose, food and water intake, GFR, and urine output gradually returned to diabetic values (Table 1). To determine the impact of reductions in food intake in mediating the chronic antidiabetic responses to leptin treatment, we also studied a group of streptozotocin-treated diabetic rats (n 4) that did not receive leptin infusion but were pair-fed the same amount of food consumed by leptin-treated rats (Fig. 1D). Pair feeding, however, caused only a modest reduction of plasma glucose from to mg/dl (Fig. 1C) that was accompanied by modest reductions in urine output, water intake, and GFR ( to ml/day, to ml/day, and to ml/min, respectively, n 4). MAP and HR in Diabetic Rats and During Chronic Intracerebroventricular or Intravenous Leptin Infusion To test whether leptin treatment would also ameliorate the cardiovascular changes associated with insulin-deficient diabetes, we measured 24 h/day MAP and HR directly using chronically implanted catheters throughout the study. STZ-induced diabetes was associated with pronounced bradycardia and decreased MAP in the vehicle-treated group ( bpm and 10 3 mmhg, respectively, at the 15th day after STZ injection, Fig. 2). Leptin treatment, intracerebroventricular or intravenous, completely reversed the fall in MAP and HR, which returned to prediabetic control values by the 10th day of leptin infusion (Fig. 2). However, intracerebroventricular leptin infusion raised HR to a greater extent compared with intravenous leptin infusion; HR increased by bpm during intracerebroventricular infusion and by bpm during intravenous infusion of leptin for 10 days. Upon cessation of leptin infusion, HR began to decrease toward the levels observed in diabetic rats before leptin infusion, with a more rapid fall observed in the intravenous leptin-infused group than in the intracerebroventricular leptin-infused group (from 370 7to bpm and from 423 9to bpm, respectively, at the 4th day after cessation of leptin infusion). Effect of 1 and 1/ 2 Adrenergic Receptor Antagonism on Metabolic, Hormonal, and Renal Responses to CNS Hyperleptinemia in Diabetic Rats To examine the role of 1 and 1/ 2 adrenergic receptor activation in mediating the chronic antidiabetic actions of leptin administered directly into the CNS, we treated the rats with a combined mixture of terazosin (an 1 adrenergic receptor antagonist) and propranolol (a 1 - and 2 -adrenergic receptor antagonist) starting 5 days before STZ injection and continuing throughout the study. We have previously shown that these doses of the antagonists block 90 95% of the MAP and HR responses to large bolus injections of phenylephrine (an -adrenergic agonist) or isoproterenol (a -adrenergic agonist) (4). Terazosin and propranolol treatment had no effect on food intake (26 1 vs g/day) or plasma glucose levels (87 4 vs mg/dl), and STZ injection caused hyperglycemia (Fig. 3A) and hyperphagia (37 3 vs g/day) in terazosin/propranolol-treated rats (n 5). Fig. 2. Responses to chronic intravenous (1.0 g kg 1 min 1 ) and intracerebroventricular (0.021 g kg 1 min 1 ) leptin or vehicle infusion on mean arterial pressure (MAP) and changes in mean arterial pressure ( MAP) and changes in heart rate, measured 24 h/day, in conscious Sprague-Dawley rats treated with streptozotocin. Data represent means SE of 5 rats in each group. Two-factor ANOVA for repeated measures with Bonferroni test for comparisons between groups and Dunnett s test for comparisons of experimental and control values within each group. Blockade of 1 and 1/ 2 adrenergic receptors did not attenuate the effects of 10 days of intracerebroventricular leptin infusion to reduce blood glucose to normal ( vs mg/dl, Fig. 3, A and B) or to reduce food intake. Water intake, urine volume, and GFR decreased to the same levels during leptin ICV infusion in rats with intact adrenergic receptors as in rats treated with adrenergic receptor antagonists (Table 2). Effects of 1 - and 1 / 2 -Adrenergic Receptor Antagonism on MAP and HR Responses to CNS Hyperleptinemia in Diabetic Rats Terazosin and propranolol treatment reduced MAP and HR by 8 1 mmhg (P 0.05) and 38 5 bpm (P 0.05), respectively, compared with control values (Fig. 3C). Combined 1 - and 1 / 2 -adrenergic receptor blockade also partially attenuated the increased HR observed during chronic intracerebroventricular leptin infusion; in diabetic rats with adrenergic blockade, HR increased by bpm after 10 days of intracerebroventricular leptin compared with bpm in rats with intact adrenergic receptors infused intracerebroventricularly with leptin (Fig. 3D). After the initial fall in MAP

5 R1279 Fig. 3. Responses to chronic intracerebroventricular (0.021 g kg 1 min 1 ) leptin infusion in vehicle-treated or in terazosin/propranolol-treated rats on blood glucose levels (A), blood glucose levels (B), heart rate (C), and heart rate (D) in streptozotocin-treated Sprague-Dawley rats. Data represent means SE of 5 rats per group. blood glucose levels and heart rate represent the absolute daily change compared with the day before leptin intracerebroventricular leptin infusion was started. Glucose and heart rate values of the intracerebroventricular leptin infusion in vehicle-treated group are the same as shown in Fig. 1C and Fig. 2, respectively. Two-factor ANOVA for repeated measures with Bonferroni test for comparisons between groups and Dunnett s test for comparisons of experimental and control values within each group. caused by terazosin/propranolol treatment, MAP remained stable throughout the experiment (83 1 and 84 2 mmhg, respectively, for the day before intracerebroventricular leptin infusion began and after 10 days of intracerebroventricular leptin infusion), suggesting that leptin also prevented the blood pressure from falling even further after diabetes was induced during 1 and 1 / 2 -adrenergic blockade. Table 2. Effects of chronic ICV (0.021 g/kg per minute) leptin infusion in rats treated with terazosin plus propranolol or treated with a 3 -selective antagonist, SR59230A, and effects of chronic 3 -activation using the selective agonist CL 316,243 on plasma insulin, renal function, and water intake in streptozotocin-induced diabetic rats Experimental Groups Plasma Insulin, U/ml GFR, ml/min Urine Volume, ml/day Water Intake, ml/day 1/ 1, 2-blockade group Control STZ - day 4 3 1* * * * STZ ICV leptin - day * * 28 14* STZ - recovery 4 1* * * * 3-blockade group control STZ - day 4 4 1* * * * STZ ICV leptin - day * * 21 5* STZ - recovery 4 1* * 75 10* 3-activation group Control STZ - day 4 4 2* * * * STZ CL316,243 - day * * * * STZ - recovery 2 1* * * * Values are presented as means SE. Values are given for day 4 of control, day 4 after STZ injection, day 10 of ICV leptin or IV CL316,243 infusion, and day 4 of recovery period. *P 0.05 compared with control.

6 R1280 Effects of 3 -Adrenergic Receptor Antagonism on Responses to CNS Hyperleptinemia in Diabetic Rats To test whether 3 -adrenergic receptor activation mediates the long-term metabolic and cardiovascular effects of CNS hyperleptinemia in diabetic rats, we treated the rats with a selective 3 -receptor antagonist, SR59230A (Sigma) during intracerebroventricular leptin infusion (n 5). Compared with control values, 3 -adrenergic receptor antagonism did not alter food intake (22 1 vs g/day), plasma glucose (94 6 vs mg/dl, Fig. 4A), plasma insulin, MAP, or HR. SR59230A treatment did not attenuate the effects of leptin to prevent the decrease in HR (Fig. 4B) or MAP (93 4 and 95 2 mmhg, respectively, for the day before intracerebroventricular leptin infusion began and after 10 days of intracerebroventricular leptin infusion) in diabetic rats. 3 -adrenergic receptor blockade also did not attenuate the ability of intracerebroventricular leptin infusion to restore euglycemia in insulin-deficient diabetic rats (Fig. 4A) or alter the effect of leptin to reduce GFR in diabetic rats (Table 2). Effects of Chronic 3 -Adrenergic Receptor Activation in Insulin-Deficient Diabetic Rats We further evaluated the role of 3 -adrenergic receptor in the regulation of glucose homeostasis by chronic infusion of a specific 3 -receptor agonist, CL316,243 (Sigma, n 4). Activation of the 3 -adrenergic receptor for 10 days in insulindeficient rats failed to have a significant antidiabetic effect (Fig. 4C). In fact, 3 -adrenergic receptor activation was associated with worsening of hyperglycemia (Fig. 4C) and hyperphagia (64 7 vs g/day for diabetic treated vs. diabetic untreated, respectively). Chronic CL316,243 infusion also further increased water intake and urine output (Table 2). No major cardiovascular or renal effects were observed during CL316,243 infusion; MAP decreased 6 1 mmhg, whereas HR decreased from 356 5to bpm in diabetic rats treated with CL316,243, similar to the changes observed in untreated diabetic rats over the same time period. GFR increased (Table 2) with the progression of the diabetes as observed in untreated diabetic rats. DISCUSSION Fig. 4. Responses to chronic intracerebroventricular leptin infusion (0.021 g kg 1 min 1 ) in vehicle-treated or in rats treated with a selective 3- adrenergic receptor antagonist, SR59230A, on blood glucose levels (A) and on heart rate (B) in streptozotocin-treated Sprague-Dawley rats. C: responses to chronic intracerebroventricular leptin infusion or intravenous infusion of a selective 3-adrenergic receptor agonist (CL316,243) on blood glucose levels in streptozotocin-treated Sprague-Dawley rats. Data represent means SE of 4 to 5 rats per group. blood glucose levels and heart rate represent the absolute change from the day before intracerebroventricular leptin infusion was started compared with the last day of leptin infusion. Glucose and heart rate values of the intracerebroventricular leptin infusion in the vehicle-treated group are the same shown in Fig. 1C and Fig. 2, respectively. Student s t-test was used for comparison between groups. In this study, we show that leptin has potent antidiabetic effects that can completely normalize plasma glucose levels in insulin-deficient diabetes. In addition, we found that long-term leptin treatment reversed the cardiovascular alterations associated with uncontrolled insulin-deficient diabetes. Furthermore, our data indicate that the chronic metabolic and cardiovascular actions of leptin can be accounted for almost entirely by direct actions on the CNS, occurring even in the absence of major changes in plasma levels of leptin and that these effects can occur independently of insulin. Finally, our data also suggest that adrenergic receptor activation contributes partially to the long-term cardiovascular effects of leptin, whereas activation 1 -, 1 -, 2 -, and 3 -adrenergic receptors, does not appear to play a major role in mediating the chronic CNS actions of leptin on glucose homeostasis in insulin-deficient diabetes. Although leptin intracerebroventricular infusion caused a small increase in plasma leptin (from 1.8 to 4.3 ng/ml), this change was within the range of normal leptin levels observed in lean rodents (30, 31) and may be caused by normalization of adipocyte function secondary to enhanced glucose utilization during intracerebroventricular leptin infusion rather than as a result of spillover from the intracerebroventricular infusion. Also, a previous study from our laboratory showed that intravenous or carotid artery infusion of leptin at a rate of 0.1 g kg 1 min 1, a rate that was 5 times as great as the intracerebroventricular leptin infusion rate used in the present study, did not raise plasma leptin levels and did not alter appetite or plasma insulin levels (29). Hidaka et al. (12) also showed that peripheral leptin injection at the same dose given intracerebroventricularly in STZ-diabetic rats resulted in a

7 twofold increase in plasma leptin concentration, but it had no effect in reducing glucose levels compared with a marked reduction in glucose levels after intracerebroventricular injection. Collectively, these data suggest that the powerful effects of leptin on glucose homeostasis are mediated mainly by leptin s direct actions on the CNS. Although leptin returned food intake to normal in insulindeficient diabetes, decreased food intake per se did not appear to play a major role in mediating the antidiabetic actions of leptin, as plasma glucose levels remained very high in diabetic rats that were pair fed the same amount of food consumed by leptin-treated rats. Other studies also suggest that the glucoselowering effects of leptin are independent of reduced food intake (12, 18). SNS activation has been suggested to be an important mediator of the CNS effects of leptin on chronic glucose homeostasis (12, 18), but this possibility has not been tested directly in previous studies. The concept that SNS activation plays a key role in mediating the effects of leptin on glucose regulation comes mainly from short-term studies in nondiabetic rats in which the increased glucose turnover and uptake by peripheral tissues, such as skeletal muscle and brown adipose tissue, during acute leptin infusion were abolished by sympathetic denervation (9, 14, 22). In the present study, we examined the potential role of the different adrenergic receptor subtypes in mediating the chronic CNS actions of leptin on glucose regulation in diabetic rats. Blockade of 1 -, 1 -, 2 -, or 3 -adrenergic receptors did not attenuate the antidiabetic effects of leptin in insulin-deficient diabetic rats. Although we did not directly test the efficacy of 3 -adrenergic receptor blockade, chronic 3 -adrenergic receptor activation also did not lower plasma glucose in diabetic rats, suggesting that 3 -adrenergic receptor activation may not play a major role in long-term regulation of glucose homeostasis, at least when insulin levels are very low. Thus, although leptin s antidiabetic effects are clearly mediated by activation of CNS pathways, our data suggest that mechanisms other than stimulation of peripheral adrenergic receptors may link the central actions of leptin with peripheral glucose regulation. In normal lean rats and in obese rats, leptin may enhance glucose uptake and metabolism, at least in part, by increasing fatty acid oxidation (19, 21). In diabetic rats, however, fatty acid oxidation is already increased and is the main source of energy (33), and leptin may not be able to further stimulate fatty acid oxidation. In fact, leptin has been shown to decrease several markers of fatty acid oxidation while restoring euglycemia in insulin-deficient diabetes (18), suggesting that leptin may cause a switch in fuel utilization from fatty acids to glucose in diabetic rats. Another possible explanation for the maintenance of leptin s potent antidiabetic effect in insulin-deficient diabetic rats during adrenergic blockade is that SNS activation may contribute to the stimulatory effect of leptin on glucose uptake by increasing insulin sensitivity, which can only occur when adequate insulin levels are present. However, we have previously shown in rats with normal insulin levels that combined - and 1 / 2 - leptin blockade did not attenuate the chronic effects of insulin on peripheral glucose utilization (4), further supporting our hypothesis that mechanisms other than adrenergic activation may link leptin s CNS effects with peripheral glucose utilization. R1281 Acute studies have shown that insulin given directly into the hypothalamus reduced liver glucose production and that this effect was mediated by parasympathetic nervous system (PNS) activation to the liver via the hepatic branch of the vagus (26). However, most of the studies demonstrating a role for the PNS on liver glucose production have been short term and whether chronic central leptin infusion activates the PNS and decreases liver glucose production remains to be determined. It is also possible that leptin may trigger the release of other CNS neurohumoral factors that could contribute to leptin s antidiabetic effects. For example, central injections of leptin or -melanocyte-stimulating hormone ( -MSH) have been shown to stimulate thyroid-releasing hormone, leading to increased plasma levels of thyroid stimulating hormone and thyroxin (T4) (15). Increased T4 levels augment glucose uptake in human mononuclear blood cells (17) and could contribute to enhanced peripheral glucose uptake during hyperleptinemia, although it is unclear whether T4 could exert the powerful antidiabetic actions seen with leptin administration. An important mediator of leptin s central actions is the hypothalamic POMC system. Leptin stimulates POMC neurons to release -MSH that activates melanocortin-4 receptors (MC4R) and appears to mediate most of the leptin s effect to suppress appetite and increase SNS activity (13, 28, 35). We have shown that blockade of MC4R abolished the chronic appetite and cardiovascular effects of leptin and attenuated the effects of leptin to reduce fasting insulin and glucose levels (6). Furthermore, MC4R-deficient mice are obese, hyperglycemic, and hyperinsulinemic, and they have a lower HR and are normotensive compared with lean wild-type mice despite pronounced hyperleptinemia (32). Conversely, activation of MC4R reduces insulin levels, while maintaining euglycemia (16) and also reduces liver glucose production (23). Whether central POMC/MC4R activation also mediates the chronic antidiabetic actions of leptin or whether it is required for leptin to fully restore heart rate and arterial pressure to control values in diabetic rats, however, remains to be determined. Arterial pressures were somewhat variable during the initial onset of diabetes, with a tendency for MAP to increase during the first 5 to 7 days after STZ injection, except in the intravenous leptin-treated group. This was followed by a significant decrease in MAP in the control group as diabetes progressed. Induction of diabetes was also associated with a progressive decline in heart rate in all groups. In the present study, we demonstrated that long-term leptin infusion completely reversed the fall in heart rate and arterial pressure and maintained them at normal values in insulin-deficient diabetic rats. Furthermore, blockade of 1 - and 1 / 2 -adrenergic receptors attenuated the increase in heart rate induced by leptin, indicating that at least part of the heart rate response to leptin in diabetic rats is mediated via adrenergic activation. The 3 adrenergic receptors, however, do not appear to play an important role in mediating the cardiovascular actions of leptin. The reduction of plasma glucose levels and the resultant attenuation of the diuresis that occur in this model may have also contributed to the normalization of blood pressure. In summary, leptin exerts a powerful antidiabetic effect in insulin-deficient diabetic rats via its direct actions on the CNS. The chronic antidiabetic effects of leptin are independent of reductions in food intake or increased peripheral 1 -, 1 -, 2 -, and 3 -adrenergic activity and can occur even in the presence

8 R1282 of nearly undetectable plasma insulin levels. Leptin also exerts important long-term cardiovascular actions that are at least partially mediated via / adrenergic activation. Although the adrenergic nervous system is important for the chronic cardiovascular actions of leptin, our data suggest that other mechanisms link the CNS actions of leptin with its powerful antidiabetic effects. Unraveling these mechanisms could provide tools for improving glycemic control in diabetic patients. ACKNOWLEDGMENTS The authors research was supported by a National Heart, Lung and Blood Institute grant PO1 HL and by AHA Southeast Affiliate Postdoctoral Fellowship Grants to Alexandre A. da Silva and Lakshmi S. Tallam. We thank Haiyan Zhang for radioimmunoassay of plasma insulin concentration. REFERENCES 1. Abe H, Minokoshi Y, and Shimazu T. Effect of beta3-adrenergic agonist, BRL35135A, on glucose uptake in rat skeletal muscle in vivo and in vivo. 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