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Endocrinology Vol. 139, No. 9 3863-3870
Copyright © 1998 by The Endocrine Society

ARTICLES

Leptin Affects Pancreatic Endocrine Functions through the Sympathetic Nervous System1

Akira Mizuno2, Takashi Murakami2, Shizuka Otani, Masamichi Kuwajima and Kenji Shima

Department of Laboratory Medicine, School of Medicine, the University of Tokushima, Tokushima 770-8503, Japan

Address all correspondence and requests for reprints to: Kenji Shima, M.D., Ph.D., Department of Laboratory Medicine, School of Medicine, the University of Tokushima, Kuramotocho 3-chome, Tokushima 770-8503, Japan. E-mail: shima{at}clin.med.tokushima-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The effects of leptin on the secretion of insulin and glucagon were examined. In an experiment involving insulin response to an iv glucose load in vagotomized rats, the plasma concentrations of insulin were significantly lower in the leptin (20 nmol/kg BW)-treated group than in a control group. However, in intact rats and rats that had undergone both vagotomy and chemical sympathectomy, this suppressive effect of leptin on insulin secretion was not detected. In an experiment involving a hypoglycemia-induced glucagon secretion test in intact rats, an iv injection of leptin (20 nmol/kg BW) augmented the plasma glucagon response to hypoglycemia. In the case of sympathectomized rats, however, this stimulative effect of leptin on glucagon secretion was not detected. In an experiment with perfused rat pancreas, the addition of leptin (20 nM) to the perfusate slightly suppressed insulin secretion, but had no effect on basal or glucopenia-induced glucagon secretion. In intact rats infused with leptin (0.31 µmol/day), the expression of uncoupling protein-1 messenger RNA in interscapular brown adipose tissue was increased, whereas no such effect of leptin on the uncoupling protein-1 messenger RNA expression was observed in brown adipose tissue in chemically sympathectomized rats. These findings suggest that leptin might indirectly affect pancreatic endocrine functions, probably through its stimulative effects on the sympathetic nervous system.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LEPTIN, the product of the obese (ob) gene, is a 16-kDa protein that is primarily produced by adipocytes (1). A recessive mutation in the ob gene causes severe hereditary obesity in the ob/ob mouse (2), and the administration of exogenous leptin reverses this obesity (3). As a result, leptin is generally thought to be involved in the control of body weight via the regulation of energy homeostasis. Other well characterized inherited obesity mutations in the mouse and rat are diabetes (db) (4) and fatty (fa) (5), which are mutations in the leptin receptor (OB-R) genes (6, 7, 8, 9).

Several alternate spliced isoforms (a–e, as well as others) of the OB-R have been cloned, and all of these, except for the OB-Re (soluble form), contain a single transmembrane domain (6, 7, 8, 9). Among them, the OB-Rb has the greatest capacity to perform signal transduction. Several lines of evidence suggest that the central nervous system, in particular the hypothalamus, is a direct target of leptin. The OB-Rb is highly expressed in those hypothalamic nuclei that are known to control the regulation of food intake and body weight (10, 11). The activity of the STAT3 transcription factor and fos gene expression are increased in the hypothalamus within 1 h after a single iv injection of leptin (12). Finally, the much higher potency in inducing weight loss of intracerebroventricular infusion of leptin than sc leptin (13) and the identification of leptin in cerebrospinal fluid are consistent with the idea that the main site of its action is in the central nervous system (14). However, the OB-Rs, including the OB-Rb, are expressed in a wide variety of tissues (7), including pancreatic islets (15, 16), in addition to the brain, and the number of reports on the extraneural action of leptin are increasing (15, 16, 17, 18, 19). Several laboratories have reported a direct action of leptin on the endocrine pancreas. However, conflicting results were observed in experiments using batch incubation or perifusion of isolated pancreatic islets, or perfused pancreas, in which leptin has an inhibitory, a stimulatory, or no effect on insulin release from the pancreatic islets (15, 16, 20, 21, 22, 23, 24, 25, 26). If leptin had a distinct direct action on the pancreatic endocrine functions, the results would be more or less alike, although they might differ slightly in the degree of effectiveness. These findings together with several reports demonstrating the stimulatory effect of leptin on sympathetic activities (27, 28, 29) prompted us to assume the possibility that it might indirectly act on pancreatic endocrine cells through the sympathetic nervous system, which has been well documented to modulate their functions (30, 31).

The synthesis of the uncoupling protein-1 (UCP1) (32), an important element in energy expenditure that exists in the adipocytes of the brown adipose tissue (BAT), is essentially regulated at the level of transcription by norepinephrine released at the surface of the adipocytes through stimulation of the sympathetic nervous system (33). Therefore, the UCP1 messenger RNA (mRNA) level in BAT represents a viable marker for the excitatory state of the sympathetic nervous system over a time course.

To ascertain whether leptin has an indirect action on pancreatic endocrine functions via the sympathetic nervous system, we examined the effects of leptin on pancreatic {alpha}- and ß-cell functions in rats that were surgically vagotomized and/or medically sympathectomized. The data show that leptin indirectly suppresses glucose-induced insulin secretion and stimulates hypoglycemia-induced glucagon secretion through activation of the sympathetic nervous system.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental design
All rats were maintained in accordance with NIH Guidelines for the Care and Use of Laboratory Animals. Male Sprague-Dawley rats, 6 weeks of age (weighing ~200 g), were purchased from Japan SLC (Hamamatsu, Japan) and used for the following experiments after acclimation for 1 week by housing them in an animal room with food and water available ad libitum.

1) The effect of leptin on insulin secretion was studied using 70 rats. Among these, a group of 30 rats that was divided into 3 groups of 10 animals each (an intact group, a group that underwent vagotomy, and a group that underwent both vagotomy and chemical sympathectomy) was subjected to an iv glucose load. Five rats from each group received a bolus injection of leptin or a control protein (see Expression and purification of leptin and control protein below; 20 nmol/kg BW) 10 min before the administration of glucose. The other 30 rats, which underwent vagotomy, were divided into 5 groups and were also subjected to an iv glucose load. Six rats of each group received a bolus injection of leptin (0, 1, 10, 20, or 40 nmol/kg BW) 10 min before the administration of glucose. The remaining 10 rats were used in an in vitro experiment. Leptin was infused continuously at a rate of 20 nM into the perfused pancreas, in which 11.1 mM glucose-induced insulin secretion was tested.

2) The effect of leptin on glucagon secretion was studied using 30 rats, which were divided into 3 groups of 10 animals each. Two groups, an intact group and a group that underwent chemical sympathectomy, were subjected to an iv injection of insulin (0.6 U/kg BW; Novo Nordisk, Bagsvaerd, Denmark) to induce hypoglycemia. Five rats from each group received a bolus injection of leptin or a control protein (20 nmol/kg BW) 10 min before the insulin injection. The remaining group was used in an in vitro experiment. Leptin was infused continuously at a rate of 20 nM into the perfused pancreas, in which glucopenia-induced glucagon secretion was tested.

3) The effect of leptin on UCP1 mRNA expression in interscapular BAT was tested using nine rats with or without chemical sympathectomy (see UCP1 expression in BAT below).

The iv glucose load test
Four days before the following tests, catheterization of the femoral vein (see below) and vagotomy (see below) were performed, whereas a chemical sympathectomy (see below) was performed 48 h before the tests. All tests were conducted while the animals were conscious. Leptin or the control protein (20 nmol/kg BW) was injected intravenously 10 min before the iv administration of D-glucose (0.5 g/kg BW). Afterward, venous blood (0.2 ml) was withdrawn from the catheter using a heparinized 1.0-ml syringe and collected into chilled tubes, at 0, 3, 6, and 30 min for the determination of plasma insulin levels and at 0, 3, 6, 30, 45 and 60 min for the determination of plasma glucose levels. In the experiment to examine the dose-dependent suppressive action of leptin on insulin secretion in the iv glucose load test (Table 1Go), several doses of leptin (0, 1, 10, 20, and 40 nmol/kg BW) were injected intravenously to vagotomized rats 10 min before the administration of D-glucose (0.5 g/kg BW). Afterward, venous blood was collected into chilled tubes at 0, 3, and 6 min for the determination of plasma insulin levels.


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  		Table 1. Plasma insulin response to an iv glucose load in vagotomized rats with the prior administration of several doses of leptin

 
Hypoglycemia-induced glucagon secretion test
Catheterization and chemical sympathectomy were carried out in the same schedule as described for the iv glucose load test. Leptin or the control protein (20 nmol/kg BW) was injected intravenously 10 min before the iv administration of regular insulin (0.6 U/kg BW; Novo Nordisk). Afterward, venous blood (0.25 ml) was withdrawn from the catheter using a heparinized 1.0-ml syringe and collected into chilled tubes containing aprotinin (50 kallikrein units; provided by Ohkura Pharmaceutical Co., Kyoto, Japan) and EDTA disodium salt (0.5 mg) at 0, 15, 30, and 45 min for the determination of plasma glucagon levels and at 0, 15, 30, 45, 60, and 90 min for the determination of plasma glucose levels.

Catheterization of the femoral vein
Under anesthesia using sodium pentobarbital (50 mg/kg BW), a silicone rubber catheter (FT-025, Bio-Medica, Osaka, Japan) was inserted into the left femoral vein, and the line of a catheter was led out through sc tissues to an IVH kit (Bio-Cannula, Bio-Medica). After the operation, the rats were kept in a special IVH cage (BG-781, Bio-Medica) and were continuously infused with physiological saline until the test.

Vagotomy
A subdiaphragmatic vagotomy was conducted by sectioning the main vagal trunk according to the method reported by Snowdon and Epstein (34) just after catheterization under anesthesia. In control rats (sham operation), laparotomy was conducted without vagotomy.

Chemical sympathectomy
6-Hydroxydopamine (6-OHDA) is known to selectively destroy sympathetic nerve terminals. According to the method described previously (35), 6-OHDA hydrobromide (Sigma Chemical Co., St. Louis, MO), which was dissolved in ice-cold 0.9% saline containing ascorbic acid (57 mM; Merck, Darmstadt, Germany), was administrated intravenously at a dose of 0.19 mmol/kg BW 48 h before the tests. The control group was given only the vehicle (0.9% saline containing 57 mM ascorbic acid). The dose of 6-OHDA used in this experiment has been shown to cause a substantial reduction in the number of adrenergic nerves in the islets of Langerhans in the mouse (36). Moreover, we confirmed that 6-OHDA administration significantly (P < 0.0001) reduced norepinephrine concentration in pancreatic tissues 48 h after the administration (6-OHDA administration, 137 ± 39 pg/g pancreatic wt; vehicle administration, 495 ± 72 pg/g pancreatic wt).

Pancreas perfusion
Pancreata from rats that had been starved for 16 h were perfused through the celiac artery via a catheter inserted into the aorta, as described previously (37). The basal medium, which was used for the perfusion, was Krebs-Ringer bicarbonate buffer (pH 7.4) supplemented with 0.2% BSA (fraction V, Sigma Chemical Co.) and 4.5% dextran (average mol wt, 71K; Sigma Chemical Co.). The pancreas was perfused at a flow rate of 2.5 ml/min with medium warmed to 37 C and equilibrated continuously with 95% O2-5% CO2. For the insulin secretion test, perfusion with the basal medium supplemented with 3.3 mM D-glucose was performed during the first 10 min, followed by perfusion with the medium supplemented with 11.1 mM D-glucose for 20 min, and then perfusion with the medium supplemented with 3.3 mM D-glucose was performed again for the final 10 min. For the glucagon secretion test, perfusion with the basal medium supplemented with 5.6 mM D-glucose was performed during the first 10 min, followed by perfusion with the medium supplemented with 1.7 mM D-glucose for 26 min. A 15-min equilibration period preceded each perfusion experiment. Leptin was dissolved at a concentration of 500 nM in the basal medium supplemented with 3.3 mM D-glucose and was infused laterally through a three-way tube at a flow rate of 0.1 ml/min. Therefore, the concentration of leptin in the perfusion medium was approximately 20 nM. Leptin or the solution without leptin (vehicle) was infused throughout the experiment. The effluent was collected at 2-min intervals in chilled tubes, which contained both aprotinin (500 kallikrein units) and EDTA disodium salt (5 mg) for the glucagon secretion test.

UCP1 expression in BAT
Four days before killing the rats, catheterization of the femoral vein was performed, whereas chemical sympathectomy was performed 48 h before killing the rats. From 24 h before killing the rats, leptin (0.31 µmol/day) or vehicle [solvent for leptin (PBS) diluted with saline] was continuously infused intravenously into the catheter using a disposable infusion pump (Surefuser-A, Nipro Co., Osaka, Japan) at a rate of 0.7 ml/h. After killing the rats, interscapular BAT was excised, and total RNAs were prepared by the guanidine thiocyanate-cesium chloride centrifugation method (38) (Fig. 2AGo) or by two extractions against Isogen (Nippon Gene Co., Toyama, Japan; Fig. 2BGo). RNAs (8 µg) were denatured in 50% formamide-2.2 M formaldehyde at 65 C for 10 min, and electrophoresed on a 1% agarose gel containing 2.2 M formaldehyde. The gel was blotted onto a Hybond-N nylon hybridization membrane (Amersham International, Aylesbury, UK). The membrane was then hybridized with the [{alpha}-32P]deoxy-CTP random priming labeled rat UCP1 complementary DNA probe (32) (provided by Dr. Daniel Ricquier, Centre National de la Recherche Scientifique, Meudon, France), washed at a stringency of 0.5 x standard saline citrate (SSC; 1 x SSC is 150 mM sodium chloride and 15 mM sodium citrate)-0.1% SDS at 65 C, and exposed to x-ray film, as described previously (39). The amount of intact RNA in each lane of the gel was judged to be constant by ethidium bromide fluorescence, which showed ribosomal RNA bands of 18S and 28S directly in the gel and after transfer of the RNA to the nylon hybridization membrane. A Bio-Image analyzer (BAS-1500Mac, Fuji Photo Film Co., Tokyo, Japan) (40) was used for quantification.



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  		Figure 2. Effects of leptin (20 nM; •) or vehicle ({circ}) on basal (3.3 mM glucose) and glucose-induced (11.1 mM glucose) insulin secretion from perfused rat pancreas. Data are expressed as the mean ± SD insulin secretion per min (n = 5 in each group with leptin or vehicle). *, P < 0.05; §, P < 0.01 (vs. vehicle).

 
Expression and purification of leptin and control protein
The rat recombinant leptin was produced in Escherichia coli using QIA expressionist (Qiagen, Hilden, Germany) in forms of NH2-terminal fusion to the His-tag sequence (the NH2-terminal amino acid sequence of the resultant fusion form is MRGS-H6-GSSRVDIEGR-mature rat leptin). The recombinant leptin was purified, refolded from inclusion bodies according to the manufacturer’s recommended protocols, and finally dialyzed against PBS. On the addition of this recombinant leptin, immediate early genes were induced in Chinese hamster ovary (CHO) cells that overexpressed the leptin receptor, but not in parent CHO cells (41), showing that our recombinant leptin specimens are bioactive.

To rule out the possibility that some impurities in our recombinant leptin might affect pancreatic functions, a control protein was used in a control experiment in place of the recombinant leptin in the experiments with the iv glucose load test and the hypoglycemia-induced glucagon secretion test. This protein was a portion of the intracellular domain (from amino acid 980 to 1129) of the rat leptin receptor (OB-Rb) (9), which was expressed, purified, refolded, and dialyzed in exactly the same manner as the recombinant leptin.

Assays
Levels of plasma glucose were determined by the glucose oxidase method using Antsense II (Bayer-Sankyo Co., Tokyo, Japan). Levels of insulin immunoreactivity were measured with a commercial kit (Eiken Chemical Co., Tokyo, Japan) with rat insulin as a standard (Novo Nordisk) (37). RIA of glucagon immunoreactivity was performed with antiserum OAL123 (Otsuka Pharmaceutical Co., Tokyo, Japan) (42), which is directed toward the C-terminal portion of glucagon and recognizes glucagon, but not glicentin or oxyntomodulin. Norepinephrine concentrations in pancreatic tissues were analyzed using the postlabel fluorogenic method with diphenylethylenediamine (Tosoh Corp., Tokyo, Japan) as described previously (43). Briefly, total pancreatic tissue from each rat was homogenized in 0.4 N perchloric acid containing 5 mM EDTA and 0.1 mM L-ascorbic acid and centrifuged at 10,000 x g for 20 min at 4 C, and the supernatant was then used for the analysis.

Calculations and statistical analysis
All results are presented as the mean ± SD. Statistical significance was determined using Student’s t test for individual comparison of means. The data presented in Table 1Go were analyzed at each time point between groups by one-way ANOVA using the StatView computer software program (Abacus Concepts, Berkeley, CA). When ANOVA showed significant differences, post-hoc analysis was performed with Scheffé’s test. Significance was accepted as P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of leptin on plasma insulin responses to an iv glucose load in untreated or treated rats
A bolus prior to injection of leptin (20 nmol/kg BW) had no significant effect on the plasma insulin or glucose response to iv glucose in intact rats, as shown in Fig. 1Go (Intact). On the contrary, leptin significantly suppressed an increase in plasma insulin concentrations while causing a rise in plasma glucose levels after an iv glucose injection in the vagotomized rats (Fig. 1Go, Vagotomy). In these rats, significant differences in plasma insulin levels at 3, 6, and 30 min were observed, and significant differences in plasma glucose levels were seen at 30, 45, and 60 min between the leptin-treated and the control protein-treated groups. However, in rats that underwent both vagotomy and sympathectomy, the effects of leptin on responses of both plasma insulin and plasma glucose to iv glucose, which were observed in vagotomized rats, were not observed (Fig. 1Go, Vagotomy and Sympathectomy). Chemical sympathectomy alone had no significant effect on the plasma insulin or glucose response to iv glucose (data not shown). As shown in Table 1Go, leptin at a dose as low as 10 nmol/kg BW, significantly (P < 0.05) suppressed the plasma insulin response to iv glucose in vagotomized rats. The suppressive effects of leptin on an increase in plasma insulin concentrations 6 min after an iv glucose load appeared to be dose dependent.



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  		Figure 1. Effects of leptin (20 nmol/kg BW; •) or the control protein ({circ}) on plasma glucose and insulin responses to an iv glucose load in intact rats (Intact), rats that underwent vagotomy (Vagotomy), or rats that underwent both vagotomy and chemical sympathectomy (Vagotomy and Sympathectomy). Data are expressed as the mean ± SD plasma glucose levels (A) or plasma insulin levels (B). Five rats were used in each group with leptin or the control protein. *, P < 0.05; #,P < 0.001 (vs. the control protein).

 
Effect of leptin on insulin secretion from perfused rat pancreas
Figure 2Go shows changes in the mean levels of insulin in the effluents from perfused rat pancreas with the perfusate supplemented with 3.3 and 11.1 mM D-glucose in the presence or absence of leptin. The rise in the D-glucose concentration provoked a biphasic stimulation of insulin secretion. In the absence of leptin, mean insulin secretion increased from a basal level of 0.9 ± 0.3 pmol/min at 10 min (just before perfusion with 11.1 mM D-glucose) to the first peak level of 4.9 ± 2.3 pmol/min at 14 min (4 min after changing to a perfusate with 11.1 mM D-glucose) and decreased gradually thereafter, with the second peak at 30 min (just before termination of perfusion with 11.1 mM D-glucose). After perfusion with 3.3 mM D-glucose, the mean levels of insulin in the effluent decreased gradually and reached the basal level at 40 min.

With leptin, the mean level of insulin in the effluents during the basal glucose condition (3.3 mM D-glucose; until 10 min) was significantly (P < 0.05) suppressed compared with that without leptin. Over the entire period after changing to perfusate with 11.1 mM D-glucose, except for near the peaks and at 40 min (10 min after changing to perfusate with 3.3 mM D-glucose; the last fraction of this experiment), the mean levels of insulin were also significantly (P < 0.05) lower with leptin than without leptin. Leptin (20 nM) suppressed the secretion of insulin from the perfused pancreas.

Effect of leptin on plasma glucagon response to insulin-induced hypoglycemia in rats with or without pretreatment with 6-OHDA
Figure 3Go shows changes in concentration of plasma glucose and plasma glucagon after an iv bolus injection of insulin (0.6 U/kg BW). After the injection of insulin to intact rats that received an injection of the control peptide 10 min before this, the plasma glucose level decreased from 4.6 ± 0.9 mM (0 min) to a minimum of 2.1 ± 0.4 mM at 30 min and then rose gradually thereafter, reaching a level of 4.3 ± 1.2 mM at 90 min, the end of the experiment (Fig. 3AGo, Intact). With the administration of leptin 10 min before the experiment (Fig. 3AGo, Intact), the plasma glucose level decreased to a minimum of 2.1 ± 0.3 mM at 15 min, similar to that in the control protein-treated rats, but increased thereafter to higher levels at 45 and 90 min compared with those in the control protein-treated rats.



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  		Figure 3. Effects of leptin (20 nmol/kg BW; •) or the control protein ({circ}) on hypoglycemia-induced glucagon secretion in intact rats (Intact) or rats that underwent chemical sympathectomy (Sympathectomy). Data are expressed as the mean ± SD plasma glucose levels (A) or plasma glucagon levels (B). Five rats were used in each group with leptin or the control protein. *, P < 0.05; §, P < 0.01 (vs. the control protein).

 
In intact rats, the plasma glucagon concentration after insulin injection was increased in both groups with and without leptin, but was higher in the leptin group than in the control protein (without leptin) group at 30 and 45 min (Fig. 3BGo, Intact). The effect of leptin on plasma glucose and plasma glucagon responses to an iv bolus insulin injection in rats pretreated with 6-OHDA are also shown in Fig. 3Go (Sympathectomy). In the rats that had been pretreated with 6-OHDA, the plasma glucose concentrations in both groups changed in the same way after insulin injection regardless of administration of leptin or the control protein (Fig. 3AGo, Sympathectomy). The same was true for changes in plasma glucagon level after insulin injection (Fig. 3BGo, Sympathectomy).

Effect of leptin on glucagon secretion from perfused rat pancreas
Figure 4Go shows changes in the mean levels of glucagon in the effluents from perfused rat pancreas with the perfusate supplemented with 5.6 and 1.7 mM D-glucose in the presence or absence of leptin. Without leptin, the mean glucagon levels increased from a basal level of 35 ± 8 fmol/min to a peak level of 219 ± 137 fmol/min 12 min after changing to the perfusate with 1.7 mM D-glucose and essentially remained at this level thereafter until it decreased, several minutes before the end of the experiment. With the addition of leptin to the perfusate, changes in mean glucagon secretion during perfusion with 1.7 mM D-glucose were quite similar to those in the experiment without leptin. Throughout the experiment, no significant differences in the corresponding glucagon secretion between the two groups were observed.



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  		Figure 4. Effects of leptin (20 nM; •) or vehicle ({circ}) on basal (5.6 mM glucose) and glucopenia-induced (1.7 mM glucose) glucagon secretion from perfused rat pancreas. Data are expressed as the mean ± SD glucagon secretion per min (n = 5 in each group with leptin or vehicle). No significant differences in corresponding glucagon secretion between the two groups were observed.

 
Effect of leptin on UCP1 mRNA expression in BAT
Consistent with the finding of Scarpace et al. (44), a continuous leptin infusion for 24 h increased the expression of the UCP1 mRNA in interscapular BAT 3- or 4-fold (Fig. 5AGo, lane 1 vs. lanes 2 and 3; Fig. 5BGo, lane 1 vs. lane 6). The injection of 6-OHDA 48 h before the excision of BAT (24 h before the start of continuous infusion of leptin) completely abolished the enhancement of UCP1 mRNA expression by leptin (Fig. 5BGo, lanes 4 and 5 vs. lane 6). 6-OHDA injection to rats without leptin infusion had no effect on the expression levels of UCP1 mRNA (Fig. 5BGo, lanes 1 vs. lane 2 and 3).



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  		Figure 5. The effect of leptin on UCP1 mRNA expression in interscapular BAT. The rat UCP1 complementary DNA probe was hybridized to electrophoresed total RNAs (8 µg) isolated from interscapular BAT excised from rats that were continuously infused intravenously with leptin [Leptin (+); 0.31 µmol/day] or vehicle [Leptin (-)] for 24 h. In B, 6-OHDA [6-OHDA (+); 0.19 mmol/kg BW] or vehicle [6-OHDA (-)] was administrated intravenously 48 h before the excision of BAT, that is 24 h before the start of continuous infusion of leptin or vehicle. The exposure period for the x-ray film was longer for B than A. Photographs of the ethidium bromide fluorescence of gels (gel) are shown with the autoradiograms. An arrowhead indicates hybridized UCP1 mRNAs. Dashes indicate the positions of 28S and 18S ribosomal RNAs. Numerals at the top indicate the expression levels of the UCP1 mRNA, quantified with a Bio-Image analyzer BAS-1500Mac, relative to the level obtained in each lane 1.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrates that rat recombinant leptin is capable of suppressing plasma insulin and augmenting plasma glucose responses to an iv glucose load in vagotomized rats. These effects of leptin on plasma insulin and glucose during an iv glucose load were not observed in intact rats or in vagotomized and chemically sympathectomized rats. According to our previous results (21) and other reports (15, 16, 22, 24), leptin may have the capacity to suppress basal insulin secretion from islets of rodents, although this matter remains controversial (16, 20, 25, 26). In the present study, leptin at a dose as high as 20 nM slightly, but significantly, suppressed insulin secretion in the perfused rat pancreas. It is unlikely, however, that the suppressive ability of leptin per se can be completely attributed to a reduced plasma insulin response to an iv glucose load and subsequent hyperglycemia in vagotomized rats. If that were the case, similar changes in plasma glucose and insulin levels would be expected in intact rats when leptin was given before the iv glucose load. The results suggest that leptin may also act indirectly on pancreatic endocrine cells. In our experiments, leptin suppressed an increase in the plasma insulin concentrations after iv administration of glucose at doses of 10 nmol/kg BW and higher. We chose 20 nmol leptin/kg BW as the dose to clarify the mechanism through which the agent affected the plasma insulin response to glucose stimulation. What are the mechanisms by which leptin suppresses the plasma insulin response to iv glucose in vagotomized rats? It has been reported that leptin stimulates sympathetic activity when given by ip (27), iv (28), or intracerebroventricular (29) injection. The present experiments confirm the finding of Scarpace et al. (44) that leptin increases the expression of UCP1 mRNA in BAT of rats. These demonstrations coupled with the reports mentioned above suggest that leptin increases sympathetic activation of the ß-adrenergic signal transduction pathway in BAT in our rats. Indeed, the stimulative action of leptin on UCP1 mRNA in BAT was abolished in chemically sympathectomized rats. The leptin- induced increase in sympathetic activity appears to take place not only in BAT but also in other target tissues, as demonstrated in reports of an increase in lumbar and renal sympathetic nerve activities caused by leptin (28, 29).

These results along with the well documented findings that alterations in sympathetic tone affect basal (30) and glucose-stimulated insulin secretion (31) suggest that leptin may modulate pancreatic endocrine functions through its stimulative activity of the sympathetic nerves ending around the pancreatic endocrine cells. It is, however, difficult to explain why leptin administration suppressed glucose-stimulated insulin secretion in vagotomized, but not in intact, rats. However, it can be reasonably presumed that leptin-induced sympathetic activity might be exaggerated in the former due to the lack of counteraction by the parasympathetic nerve, whereas it might be counterbalanced in the latter by activation of a parasympathetic system, which per se augments glucose-stimulated insulin secretion. Hisatomi et al. (45) reported that the depletion of norepinephrine from sympathetic nerve endings by treatment with 6-OHDA lowered the pancreatic norepinephrine content, which was confirmed in our experiments. Therefore, the leptin-induced suppression of insulin secretion would be expected to be abolished in rats that underwent both vagotomy and chemical sympathectomy. This was, in fact, observed, thus supporting the idea that leptin-induced suppression of insulin secretion is mediated through its adrenergic stimulation. However, we found no significant effect of the pretreatment with 6-OHDA on plasma insulin and glucose responses to iv glucose. It can be assumed that the chemical sympathectomy alone could not enhance insulin secretion in response to hyperglycemia.

To our knowledge, this study represents the first investigation of the effect of leptin on glucopenia-induced glucagon secretion from the perfused rat pancreas, although its effect on the glucose-induced suppression of glucagon secretion has been reported (20, 26). Leptin, when tested at a concentration of 20 nM, failed to affect the stimulative action of glucopenia on glucagon secretion from the perfused rat pancreas. Moreover, we were unable to detect any significant effect of 80 nM leptin on glucopenia-induced glucagon secretion from cultured pancreatic islets (unpublished observation). Judging from our data together with Leclercq-Meyer’s finding (20, 26) of the failure of 10 nM leptin to counteract the effects of glucose on glucagon secretion in the perfused rat pancreas, it is likely that leptin per se, at the dose used here, does not significantly affect glucagon secretion from pancreatic {alpha}-cells. It has been reported (45) that in the isolated rat pancreas, adrenergic mediation accounts for most of the glucagon response to glucopenia, which is controlled within the pancreas, possibly through a direct enhancement by glucopenia of norepinephrine release from nerve endings. Hypoglycemia-induced glucagon secretion has also been reported to be accompanied by pancreatic norepinephrine spillover in the conscious dog (46), suggesting that neural signaling to pancreatic {alpha}-cells is responsive to changes in the glucose level. Leptin administration may increase glucopenia-induced norepinephrine release from nerve endings and bring about an augmented plasma glucagon response to hypoglycemia in intact rats. The fact that the leptin-induced augmentation of the plasma glucagon response to hypoglycemia was abolished in rats that had been treated with 6-OHDA is compatible with the idea that the effects of leptin on glucagon secretion are mediated by the autonomic nervous system. The findings of others appear to be inconsistent with ours, in that leptin affected the levels of neither plasma glucose, insulin, nor glucagon in intact animals when given by iv (28, 47) or intracerebroventricular (29, 47) injection. However, this is not the case. In our experiments, no effect of leptin on insulin secretion were detected in intact rats. The suppressive effect of leptin on insulin secretion and the augmented effect on plasma glucose levels were observed only after an iv glucose load into vagotomized rats. Furthermore, the stimulative effect of leptin on glucagon secretion was detected only in response to hypoglycemia.

At present, we have no data on the sites where iv administrated leptin acts, i.e. the central nervous system or nerve endings in the pancreas. The findings that intracerebroventricular administration of leptin stimulated sympathetic activity (29) and that the OB-Rb, which performs the highest signaling activities among the leptin receptors, are most abundant in the brain, especially the hypothalamus (10, 11), suggest that the observed effects of peripheral leptin on pancreatic endocrine functions are probably the result of transport into the central nervous system by a specific transport process (48, 49) and subsequent interaction with receptors there. However, the possibility cannot be excluded that the iv administrated leptin activates afferent nerve terminals, as reported by Wang et al. (50), which transmit signals to the central nervous system, resulting in stimulation of the sympathetic nerves.

In summary, iv administration of leptin suppressed plasma insulin and augmented plasma glucose responses to an iv glucose load in vagotomized rats, whereas these effects of leptin were not observed in intact rats or rats that underwent both vagotomy and chemical sympathectomy. An iv administration of leptin augmented the plasma glucagon response to insulin-induced hypoglycemia in intact rats. However, this effect of leptin on pancreatic {alpha}-cells was absent in chemically sympathectomized rats. In an experiment with perfused rat pancreas, leptin showed a suppressive effect on insulin secretion, but showed no significant effect on glucopenia-induced glucagon secretion. An iv infusion of leptin augmented UCP1 mRNA expression in BAT in the intact rat, whereas no such effect of leptin on UCP1 mRNA expression was observed in BAT in chemically sympathectomized rats. These findings suggest that leptin, when administrated intravenously, affects pancreatic endocrine functions, probably through its stimulatory effect on the sympathetic nervous system.


   Acknowledgments
 
We thank Dr. Masako Sei for the statistical analysis, Ms. Izumi Sato for technical assistance, Dr. Toshihiko Yanagida (Miyazaki Medical College) for his valuable advice, and SRL (Tokyo, Japan) for measurement of norepinephrine concentrations in pancreatic tissues from rats.


   Footnotes
 
1 This work was supported by a research grant from the Ministry of Education and Culture of Japan (to K.S.) and grants from the T. Nanba Memorial Health Care Foundation and the Clinical Pathology Research Foundation of Japan (to T.M.). Back

2 Both authors contributed equally to this work. Back

Received January 22, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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