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Endocrinology Vol. 149, No. 2 445-454
Copyright © 2008 by The Endocrine Society

Long-Term Effects of Central Leptin and Resistin on Body Weight, Insulin Resistance, and β-Cell Function and Mass by the Modulation of Hypothalamic Leptin and Insulin Signaling

Sunmin Park, Sang Mee Hong, So Ra Sung and Hye Kyung Jung

Department of Food and Nutrition, College of Natural Science, Institutes of Basic Science, Hoseo University, Asan-Si, Chungnam-Do 336-795, Korea

Address all correspondence and requests for reprints to: Sunmin Park, Ph.D., Department of Food and Nutrition, Hoseo University, 165 Sechul-Ri Baebang-Myun Asan-Si, Chungnam-Do 336-795, Korea. E-mail: smpark{at}hoseo.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To determine the long-term effect of central leptin and resistin on energy homeostasis, peripheral insulin resistance, and β-cell function and mass, intracerebroventricular (ICV) infusion of leptin (3 ng/h), resistin (80 ng/h), leptin plus resistin, and cerebrospinal fluid (control) was conducted by means of an osmotic pump for 4 wk on normal rats and 90% pancreatectomized diabetic rats fed 40% fat-energy diets. Overall, the effects were greater in diabetic rats than normal rats. Leptin infusion, causing a significant reduction in food intake, decreased body weight and epididymal fat. However, resistin and leptin plus resistin reduced epididymal fat with decreased serum leptin levels in comparison with the control. Unlike serum leptin, only resistin infusion lowered serum resistin levels. Central leptin increased glucose infusion rates during euglycemic hyperinsulinemic clamp and suppressed hepatic glucose production in the hyperinsulinemic state in comparison with the control. However, central leptin did not affect glucose-stimulated insulin secretion and β-cell mass. Central resistin infusion also increased peripheral insulin sensitivity, but not as much as leptin. Unlike leptin, resistin significantly increased first-phase insulin secretion during hyperglycemic clamp and β-cell mass by augmenting β-cell proliferation. These metabolic changes were associated with hypothalamic leptin and insulin signaling. ICV infusion of leptin potentiated signal transducer and activator of transcription 3 phosphorylation and attenuated AMP kinase in the hypothalamus, but resistin had less potent effects than leptin. Leptin enhanced insulin signaling by potentiating IRS2->Akt pathways, whereas resistin activated Akt without augmenting insulin receptor substrate 2 phosphorylation. In conclusion, long-term ICV infusion of leptin and resistin independently improved energy and glucose homeostasis by modulating in different ways hypothalamic leptin and insulin signaling.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INCREASED ADIPOSE MASS has been known to cause the development of insulin resistance and type 2 diabetes mellitus. This is related to adipocyte-secreted signaling molecules that affect glucose homeostasis such as fatty acids, adiponectin, leptin, IL-6, TNF-{alpha}, and resistin. Among these hormones, leptin and resistin have an important role in regulating weight and glucose metabolism (1). They work in similar and opposite ways, and they also interact with each other. Circulating levels of leptin and resistin are elevated in rodent models of obesity and rodents fed a high-fat diet (2, 3). In addition, leptin and resistin are similarly regulated by nutritional status; they are reduced by fasting and increased by feeding (3, 4). This effect is mediated in part through insulin and glucose (3, 4).

By contrast, there are some discrepancies in metabolic regulation between leptin and resistin. Leptin-deficient ob/ob mice develop early-onset obesity, severe insulin resistance, steatosis, neuroendocrine deficits, and diabetes (2). In addition, a daily ip injection of leptin inhibits food intake, reduces body weight and fat mass, and normalizes glycemia by promoting insulin action in ob/ob mice. In contrast to leptin, resistin knockout mice and transgenic mice expressing dominant-negative resistin in adipose tissues improved glucose tolerance and insulin sensitivity with increased adipogenesis (5). The administration of resistin impaired glucose tolerance, and mice lacking resistin decreased their overnight fasted serum glucose levels due to reduced hepatic glucose production (6, 7, 8). Rosiglitazone, insulin-sensitizing agents, decreased serum resistin levels, whereas they increased or maintained serum leptin levels in different strains of rodents (9). In addition, some studies have demonstrated a functional link between leptin and resistin by providing evidence that leptin treatment decreased resistin mRNA expression and protein levels in ob/ob mice (8, 10), and loss of resistin improved glucose homeostasis in leptin deficiency (11).

Leptin is known to act through the central nervous system to reduce food intake by regulating neuropeptides in the hypothalamus such as the inhibition of neuropeptide Y and agouti-related protein (as an orexigenic factor) and causing the stimulation of proopiomelanocortin (as an anorexigenic factor) (12). In addition, leptin acts as a signal of peripheral nutritional status to the hypothalamus, thereby modulating neurotransmitter systems that regulate energy expenditure and glucose homeostasis (10, 13). The presence of leptin is involved in the activation of leptin signaling in the hypothalamus, resulting in increased sympathetic stimulation of peripheral tissues (14). Long-term leptin infusion to the hypothalamus altered the signal transducer and activator of the transcription 3 (STAT3) signaling pathway that may be involved in food intake and glucose regulation in rodents. Its infusion also activates the sympathetic nervous system to increase energy expenditure by augmenting the resting metabolic rate (12, 15).

However, it is unknown whether resistin regulates peripheral glucose homeostasis through the alteration of hypothalamic signaling or whether leptin and resistin have a functional link in the hypothalamus. To determine whether the elevation of leptin and/or resistin in the hypothalamus plays a significant role in the progression of the diabetic status, we tested whether intracerebroventricular (ICV) leptin and/or resistin infusion modulated leptin and insulin signaling in the hypothalamus and/or regulated peripheral insulin sensitivity and pancreatic β-cell function and mass in diabetic rats fed a high-fat diet. To determine whether the effects of central leptin and resistin were confined to diabetic rats, experiments were conducted in normal sham-operated rats.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and diets
Male Sprague-Dawley rats weighing 219 ± 12 g were housed individually in stainless steel cages in a controlled environment (23 C and a 12-h light, 12-h dark cycle) with unrestricted access to food and water. All rats consumed a high-fat diet to determine the effects of central leptin and resistin on an insulin-resistant state. A high-fat diet was based on a semi-purified diet, which was modified on the AIN-93 formulation for experimental diets (16). The diet consisted of 38 energy percent (En%) carbohydrates, 22 En% protein, and 40 En% fats. The sources of carbohydrates, protein, and fats were starch, casein, and shortening, respectively. All surgical and experimental procedures were performed in accordance with the recommendations found in the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health, and they were approved by the Institutional Animal Care and Use Committee of Hoseo University, Korea.

Surgical procedures
The rats had a 90% pancreatectomy (Px) using the Hosokawa technique and sham operation (17). After 1 wk of recovery, the Px rats showed the characteristics of mild type 2 diabetes mellitus with insulin deficiency and normal body weight. They had random fed serum glucose levels of 9.4–11.8 mM, and their insulin secretion capacity was lower than sham-operated normal rats by about 50%. Px rats are well documented as showing mild type 2 diabetes with insulin deficiency and insulin resistance in many studies, including our own (17, 18, 19). Sham rats did not show any diabetic symptoms.

The rats recovered from the sham operation and the Px rats were anesthetized with ketamine and xylazine (100 and 10 mg/kg body weight, respectively) and placed in a stereotaxic device with a midline incision of the scalp exposing the periosteum. The rats were implanted with a stainless steel cannula (22-gauge) to connect an osmotic pump in the right lateral ventricle stereotaxically, using the following coordinates: 0.8 mm posterior, 1.6 mm lateral, 3.7 mm ventral to bregma (20). After the cannula was secured with dental cement, it was connected to 22-gauge tubing filled with a designated solution.

Experimental design and metabolic analysis
After 1 wk of recovery from the implantation of the cannula into the lateral ventricle, the Px and sham rats with the implantation were divided into four different groups, and they were provided the assigned hormones via osmotic pump (Alzet Osmotic Pump Co., Cupertino, CA) containing 1) cerebrospinal fluid (CSF, as a control), 2) leptin, 3) resistin, and 4) leptin plus rat resistin. The osmotic pump provided CSF, 3 ng recombinant rat leptin (PeproTech Inc., Rocky Hill, NJ), 80 ng recombinant murine resistin (PeproTech Inc.), or 3 ng leptin plus 80 ng resistin into the lateral ventricle for 4 wk on an hourly basis. The dosage of leptin and resistin infusion was assigned proportionately according to the serum levels of normal rats and the maximization of its effect on β-cell function in our cell-based preliminary study.

Overnight fasted serum glucose levels, food and water intake, and body weight were measured every Tuesday at 1000 h. An oral glucose tolerance test (OGTT) was performed every week in overnight fasted rats by orally administering 2 g/kg glucose so as to use it as a marker for the potency of leptin and resistin. The results were not significantly different during the first, second, and third weeks, which indirectly revealed no signs of changes in hormonal function during the experiment (data not shown). Serum glucose and insulin were measured at 0, 10, 20, 30, 45, 60, 90, and 120 min after glucose loading by bleeding the tail, and the average area under the curve of glucose and insulin was calculated. Serum glucose levels were analyzed with a Glucose Analyzer II (Beckman, Palo Alto, CA), and serum insulin, leptin, and resistin levels were measured by commercial RIA kit (Linco Research, St. Charles, MO).

Glucose-stimulated insulin secretion
After the catheterization of the right carotid artery and left jugular vein at the third week from infusing ICV leptin or resistin, a hyperglycemic clamp was performed in free-moving and overnight fasted rats to determine insulin secretion capacity as described in previous studies (19, 21). During the clamp, glucose was infused to maintain serum glucose levels of 5.5 mM above baseline, and serum insulin levels were measured at designated times. After the clamp, rats were freely provided with food and water for 2 d, and the next day, they were deprived of food for 16 h. The rats were anesthetized with the mixture of ketamine and xylazine, and 100 nM insulin was injected through the inferior vena cava of the rats. Fifteen minutes later, they were killed by decapitation, and tissues were rapidly collected, frozen in liquid nitrogen, and stored at –70 C to determine leptin and insulin signaling.

Insulin resistance
After 3 wk of treatment, catheters were surgically implanted into the right carotid artery and left jugular vein of rats anesthetized with ip injections of ketamine and xylazine (100 and 10 mg/kg body weight, respectively). After 5–6 d of implantation, a euglycemic-hyperinsulinemic clamp was performed on fasted conscious rats to determine insulin resistance as previously described (19, 22). [3-3H]Glucose (NEN Life Science Products Life Science, Boston, MA) was continuously infused during a 4-h period at the rate of 0.05 µCi/min. Basal hepatic glucose output was measured in blood collected at 100 and 120 min after initiation of the [3-3H]glucose infusion. Then, a primed continuous infusion of human regular insulin (Humulin; Eli Lilly, Indianapolis, IN) was initiated at a rate of 20 pmol/kg·min to raise plasma insulin concentration to approximately 1100 pM. Glucose (25%) was infused at variable rates as needed to clamp glucose levels at about 6 mM. Although blood glucose levels were steady between 200 and 240 min, the rate of glucose production was determined by measuring every 10 min the blood levels of [3-3H]glucose and 3H2O. Clamped hepatic glucose output was calculated by subtracting glucose infusion rates (GIR) from the rates of glucose appearance. Whole-body glucose disposal was expressed in terms of milligrams of glucose per kilogram of body weight per minute required to maintain euglycemia during hyperinsulinemia (19, 22). At the end of the clamp, the rats were anesthetized with sodium pentobarbital (35 mg/kg body weight) (Nembutal; Abbott Laboratories, North Chicago, IL) and were killed by decapitation. Tissues were rapidly dissected, weighed, and frozen in liquid nitrogen, and stored at –70 C until further analysis could be performed. Serum glucose levels were analyzed with a Glucose Analyzer II (Beckman). Serum insulin and leptin levels were measured by RIA (Linco Research, St. Charles, MO).

Immunohistochemistry and islet morphometry
Six rats from each group were treated with 5-bromo-2-deoxyuridine (BrdU; Roche Molecular Biochemicals, Indianapolis, IN; 100 µg /kg body weight) at the end of the 12-wk experimental period. Six hours after injection, pancreas samples were prepared and analyzed as previously described (23). The pancreas was dissected, fixed in a 4% paraformaldehyde solution (pH 7.2) overnight at room temperature, and embedded in paraffin blocks. Serial 5-µm paraffin-embedded tissue sections were mounted on slides. To prevent the selection of sections with the same islet twice, after rehydration, every sixth or seventh section was selected to determine β-cell area, BrdU incorporation, and apoptosis. The randomly chosen sections were immunostained as previously described (23).

Endocrine β-cells were identified by applying a guinea pig antiinsulin antibody in paraffin-embedded pancreatic sections. β-Cell proliferation was examined by the incorporation of BrdU in β-cells from rats injected with BrdU. This incorporation was determined by performing a double-label immunohistochemistry with anti-insulin (Zymed Laboratories, South San Francisco, CA) and anti-BrdU antibodies (Roche, Mannheim, Germany) on rehydrated paraffin sections. Apoptosis of β-cells was measured by TUNEL kit (Roche) in paraffin sections of the pancreas and counterstained with hematoxylin and eosin to visualize islets (19). Quantification of β-cell area, BrdU+ cells, and apoptotic bodies in islets was performed as explained in our previous study (19, 23).

Immunoblot analysis
The hypothalamus was lysed in 20 mM Tris buffer (pH 7.4) containing 2 mM EDTA, 137 mM NaCl, 1% Nonidet P-40, 10% glycerol, and 12 mM {alpha}-glycerol phosphate and protease inhibitors. After 30 min on ice, the lysates were centrifuged for 10 min at 12,000 rpm at 4 C. After measuring protein contents in lysate by Bio-Rad protein assay kit, lysate with equal amounts of protein were immunoprecipitated with specific antibodies before or directly resolved by SDS-PAGE antibodies against insulin receptor substrates (IRS)-2 (UBI, Waltham, MA), protein kinase B (or Akt; Cell Signaling Technology, Beverly, MA), phosphorylated protein kinase BSer473 (Cell Signaling Technology), STAT3 (Cell Signaling Technology), phosphorylated STAT3Tyr705, AMP kinase (AMPK; Cell Signaling Technology), and phosphorylated AMPKThr172 and β-actin (Santa Cruz Biotech) were used, as previously described (19, 23). The intensity of protein expression was determined using Imagequant TL (Amersham Biosciences, Piscataway, NJ). These experiments were repeated three times for each group.

Statistical analysis
All results are expressed as a mean ± SD. Statistical analysis was performed using the SAS statistical analysis program. The significance of leptin and resistin effects was determined by one-way ANOVA. Differences among groups with a P < 0.05 were considered statistically significant by Tukey test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The chronological changes in body weight and food intake
To assess closely the time course for the metabolic actions of ICV leptin and resistin in normal and diabetic rats, body weight, food intake, and overnight fasted serum glucose and insulin levels were evaluated for 4 wk and presented in Fig. 1Go. Food intake showed an initial dramatic decrease in leptin-infused rats for 4 d in diabetic rats, and after that, it gradually increased, but it remained significantly lower in diabetic rats compared with that of the control group (Fig. 1Go). The food intake of normal rats exhibited a similar pattern to diabetic rats (Tables 1Go and 2Go). Unlike leptin, ICV resistin infusion did not cause any significant difference in food intake, compared with CSF infusion in normal and diabetic rats. Furthermore, resistin blocked the leptin effect on food intake in the ICV infusion of leptin plus resistin in normal and diabetic rats (Fig. 1Go and Tables 1Go and 2Go).


Figure 1
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  		FIG. 1. Effect of central CSF (vehicle), leptin, resistin, and leptin plus resistin on food intake and body weight in Px diabetic rats. During the experimental periods of ICV infusion of CSF, leptin, resistin, and leptin plus resistin (Lep+Res), food intake (A) and body weight (B) were measured weekly in Px diabetic rats on Tuesdays at 1000 h after overnight fasting. The sample size in each group was the same as Tables 1Go and 2Go in normal and diabetic rats, respectively. Values are means ± SD. *, The leptin group was significantly different from other groups at P < 0.05; **, P < 0.01.

 

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  		TABLE 1. Epididymal fat weight and overnight fasted serum glucose, insulin, leptin, and resistin levels in normal sham rats at the end of experimental period

 

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  		TABLE 2. Epididymal fat weight and overnight fasted serum glucose, insulin, leptin, and resistin levels in diabetic rats at the end of experimental period

 
Body weight progressively increased throughout the infusion periods in normal and diabetic rats infused with CSF (Tables 1Go and 2Go). In leptin-infused diabetic rats, body weight remained at similar levels from d 0 for 2 wk, and then it gradually increased in diabetic rats, although it remained lower than the control group (Fig. 1Go). Central resistin infusion increased body weight in a similar way to the CSF-infused diabetic rats, and consistent with food intake, resistin nullified the leptin effect on body weight (Fig. 1Go). Overall, the changes in body weight in normal rats exhibited similar patterns to those in diabetic rats (Tables 1Go and 2Go). However, resistin decreased body weight as much as leptin in diabetic rats, but not in diabetic rats. Epididymal fat mass, representing visceral fat contents, markedly decreased in leptin-infused rats in normal and diabetic rats, compared with the CSF-infused rats (Tables 1Go and 2Go). ICV infusion of resistin and leptin plus resistin somewhat decreased the fat mass in normal and diabetic rats, but the reduction did not reach the leptin infusion level (Tables 1Go and 2Go). Thus, chronic ICV infusion of leptin and resistin decreased body fat (accompanied by lower food intake) in the early stage of the treatment in normal and diabetic rats.

Overnight fasted serum glucose, insulin, leptin, and resistin levels
In comparison with overnight fasted serum glucose and insulin levels between normal and diabetic rats, we noticed that Px diabetic rats had insulin deficiency. Overnight fasted serum glucose levels exhibited no difference among all groups of normal and diabetic rats (Tables 1Go and 2Go). However, central infusion of leptin, resistin, or leptin plus resistin decreased overnight-fasted serum insulin levels in normal and diabetic rats compared with the control group (Tables 1Go and 2Go). In parallel with the amount of epididymal fat pads, central leptin and resistin decreased serum leptin levels in normal and diabetic rats, whereas central leptin reduced them the most in diabetic rats. The serum leptin levels among the groups reflected decreased body fat. Circulating levels of resistin decreased with the central infusion of resistin in normal and diabetic rats, whereas they were not altered by the central infusion of leptin (Tables 1Go and 2Go). Serum leptin and resistin levels did not show any indication of leakage of centrally infused leptin and resistin (Tables 1Go and 2Go).

Glucose tolerance during OGTT
During the third week of administration, ICV infusion of leptin, resistin, and leptin plus resistin lowered serum glucose levels from their peak in sham and diabetic rats, and the infusion quickly reduced the levels in a comparable manner during OGTT compared with the CSF infusion (Fig. 2Go, A and B). Serum glucose levels were higher in diabetic rats by approximately 57% compared with normal sham rats during the OGTT. This was explained by reduced serum insulin levels of about 96%. However, their serum insulin levels exhibited different patterns from serum glucose in normal and diabetic rats during OGTT. Serum insulin levels were comparable in all groups at 40 and 60 min during OGTT in normal and diabetic rats, whereas at 90 and 120 min, they were higher only in the CSF group compared with the other groups (Fig. 2Go, C and D). The area under the curve of serum glucose in OGTT decreased in normal and diabetic rats infused with leptin, resistin, and leptin plus resistin compared with CSF-infused rats (Fig. 2Go, E and F). However, the area under the curve of serum insulin was not significantly different among all groups in the normal and diabetic rat categories (Fig. 2Go, E and F).


Figure 2
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  		FIG. 2. The changes of serum glucose and insulin levels during OGTT. At the third week of ICV infusion of CSF, leptin, resistin, and leptin plus resistin (Lep+Res), OGTT was performed in overnight fasted rats by orally administering 2 g/kg glucose. Serum glucose (A and B) and insulin (C and D) were measured at 0, 10, 20, 30, 45, 60, 90, and 120 min after glucose loading by tail bleeding, and the average of area under the curve of glucose and insulin (E and F) was calculated in normal and diabetic rats, respectively. The sample size in each group was the same as Tables 1Go and 2Go in normal and diabetic rats, respectively. Values are means ± SD. *, The leptin group was significantly different from other groups at P < 0.05; **, P < 0.01; ***, P < 0.001; a and b, values on the same parameter with different letters were significantly different at P < 0.05.

 
Insulin secretion during hyperglycemic clamp
To characterize β-cell function in rats centrally infused with leptin, resistin, and leptin plus resistin, glucose-stimulated insulin secretion was measured by hyperglycemic clamp. During hyperglycemic clamp, serum insulin levels peaked at 2–5 min and then declined to a nadir at 10 min, called first-phase insulin secretion; they were elevated again and reached a maximum at 60–120 min, called second-phase insulin secretion (Fig. 3Go). Overall, insulin secretion capacity, represented by serum insulin levels in first and second phases, was higher by about twice as much in normal rats than diabetic rats (Tables 3Go and 4Go). ICV leptin suppressed first-phase insulin secretion in normal and diabetic rats, whereas ICV resistin stimulated it (Fig. 3Go, A and B). In the central infusion procedure of leptin plus resistin, leptin nullified the resistin effect on first-phase insulin secretion. By contrast, second-phase insulin secretion was higher in the control group of diabetic rats than the other groups, whereas it was not significantly different among the treatments administered to normal rats (Fig. 3Go, A and B). This indicated that β-cells had sufficient insulin secretion capacity to secrete during the first-phase insulin section and tightly regulated second-phase insulin secretion in normal rats. However, β-cells did not contain adequate capacity and impaired the regulation in diabetic rats. Resistin prevented the loss of β-cell function especially in diabetic rats. Due to increased second-phase insulin secretion, especially from 10–90 min, the area under the curve of serum insulin was greater in the control group in diabetic rats than the other groups, but the area was not significantly different with regard to the treatments administered to normal rats (Tables 3Go and 4Go).


Figure 3
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  		FIG. 3. The changes of serum insulin levels during hyperglycemic clamp. At the end of experimental periods of ICV infusion of CSF, leptin, resistin, and leptin plus resistin (Lep+Res), a hyperglycemic clamp to maintain blood glucose levels 100 mg/dl above fasting levels was performed in overnight fasted normal (A) and diabetic (B) rats to determine insulin secretion patterns and capacity at the end of experimental periods. Sample size of each group was the same as in Tables 3Go and 4Go in normal and diabetic rats, respectively. Values are means ± SD. *, The control (CSF infusion) group was significantly different from other groups at P < 0.05.

 

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  		TABLE 3. Insulin secretion capacity during hyperglycemic clamp in normal rats

 

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  		TABLE 4. Insulin secretion capacity during hyperglycemic clamp in diabetic rats

 
Because GIR during hyperglycemic clamp indicated β-cell function and insulin sensitivity at a hyperglycemic state, calculated as the ratio of GIR to steady-state serum insulin levels (24), GIR and insulin sensitivity at hyperglycemic states were higher in normal rats than diabetic rats (Tables 3Go and 4Go). GIR to maintain serum glucose levels 5.5 mM above the baseline was found to be greater in the ascending order of leptin, leptin plus resistin, resistin, and CSF in normal sham rats and diabetic Px rats (Tables 3Go and 4Go). The insulin sensitivity during the hyperglycemic state showed the comparable pattern of GIR during hyperglycemic clamp in all rats (Tables 3Go and 4Go). Therefore, the results of hyperglycemic clamp revealed that ICV leptin and resistin improved glucose homeostasis by independent pathways during hyperglycemic states; ICV leptin enhanced it by improving insulin sensitivity without modulating insulin secretion capacity, whereas ICV resistin improved it by potentiating insulin secretion capacity.

Insulin resistance during euglycemic hyperinsulinemic clamp
To determine to what extent improved insulin sensitivity had been increased, a euglycemic-hyperinsulinemic clamp was conducted. As predicted from body weight and serum glucose and insulin levels, the exogenous GIR required to maintain euglycemia was markedly increased in the leptin group. GIR was higher in normal rats than diabetic rats. Central leptin and resistin raised GIR in normal and diabetic rats, but the incremental effect was greater in diabetic rats than normal rats (Fig. 4Go, A and B). Only in diabetic rats did ICV resistin infusion increase GIR less than leptin. The infusion of leptin plus resistin did not reach the same level as leptin, which was possibly due to the blocking effect of resistin. This suggests that resistin offsets the leptin effect on insulin sensitivity (Fig. 4Go, A and B). Unlike GIR, neither leptin nor resistin improved insulin-simulated whole-body glucose uptake in normal and diabetic rats (Fig. 4Go, A and B).


Figure 4
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  		FIG. 4. Glucose disposal rates, glucose uptake, and hepatic glucose output during the basal state and clamped state during euglycemic hyperinsulinemic clamp. At the end of experimental periods of ICV infusion of CSF, leptin, resistin, and leptin plus resistin (Lep+Res), a euglycemic-hyperinsulinemic clamp was performed in overnight fasted normal and Px diabetic rats to determine whole-body glucose disposal rates and glucose uptake (A and B) and hepatic glucose output at basal and clamped states at 1100 pM serum insulin (C and D). The sample size in each group was the same as Tables 3Go and 4Go in normal and diabetic rats, respectively. Values are means ± SD. *, There were significant differences among the groups at P < 0.05; a–c, values on the same parameter with different letters were significantly different at P < 0.05.

 
Hepatic glucose output in the basal (fasting) and hyperinsulinemic states was higher in diabetic rats than normal rats during hyperinsulinemic-euglycemic clamp (Fig. 4Go, C and D). However, central infusion of leptin and resistin did not significantly alter hepatic glucose output in the basal state, which was comparable to overnight fasted serum glucose levels (Fig. 4Go, C and D). A good portion of the difference in the GIR likely resulted from the reduction of the insulin-stimulated hepatic glucose production at a hyperinsulinemic state in normal and diabetic rats (Fig. 4Go, C and D), which represents the attenuation of hepatic insulin sensitivity. During a hyperinsulinemic clamped state, at approximately 1100 pM serum insulin levels, leptin exhibited the lowest hepatic glucose output in normal and diabetic rats (Fig. 4Go, C and D), indicating that ICV leptin enhanced hepatic insulin sensitivity. ICV resistin also decreased hepatic glucose output in hyperinsulinemic clamped states in normal and diabetic rats, but leptin suppressed the output more than resistin. In addition, leptin plus resistin did not reduce the hepatic glucose production up to the leptin infusion level, suggesting that resistin somewhat nullified the leptin action in hepatic glucose output, and leptin and resistin worked in independent pathways (Fig. 4Go, C and D).

Pancreatic β-cell mass, proliferation, and apoptosis
The percentage of β-cell area in the total pancreas area of a section was less in normal rats than diabetic rats because β-cell growth was much greater in normal rats via proliferation and neogenesis after the removal of the pancreas in diabetic Px rats (Tables 5Go and 6Go). Resistin infusion significantly increased β-cell area in normal and diabetic rats, whereas leptin infusion reduced the area in diabetic rats only (Tables 5Go and 6Go). Infusion of leptin plus resistin increased the area in normal and diabetic rats, but not as much as resistin by itself, suggesting that leptin counteracted the resistin effect on β-cell growth in diabetic Px rats. At the end of the experiment, diabetic Px rats had a smaller pancreas than normal rats by 51.4 ± 7.5% instead of 90% because the pancreas was regenerated after the removal of 90% of the pancreas. Leptin or resistin infusion did not affect pancreas weight in normal and diabetic rats (data not shown). Pancreatic β-cell mass, calculated by multiplying β-cell area by the pancreas weight, exhibited the same patterns of β-cell area. The differences of β-cell mass among the groups depended on individual cell size and β-cell proliferation and apoptosis. ICV infusion of leptin, resistin, and leptin plus resistin decreased the individual β-cell size in diabetic rats compared with the CSF-infused control rats (Tables 5Go and 6Go). In normal rats, leptin decreased the individual β-cell size but resistin and leptin plus resistin did not reduce individual size significantly.


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  		TABLE 5. The modulation of islet morphometry in normal rats

 

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  		TABLE 6. The modulation of islet morphometry in diabetic rats

 
With regard to β-cell growth and survival, only resistin stimulated β-cell proliferation in normal and diabetic rats whereas leptin did not modulate it (Tables 5Go and 6Go). The resistin effect on the proliferation was nullified with leptin in normal rats but not in diabetic rats. Apoptosis increased leptin infusion the most in diabetic rats only, whereas resistin infusion reduced it the most. Apoptosis was not significantly affected by central leptin and resistin in normal rats (Table 5Go). Because leptin and resistin completely blocked their effects in diabetic rats, leptin plus resistin exhibited the median of leptin and resistin. As a result, ICV infusion of resistin increased the number of β-cells (hyperplasia) by enhancing proliferation and reducing apoptosis in normal and diabetic Px rats, whereas leptin decreased the number by potentiating apoptosis. Hyperplasia occurred in normal rats with a smaller portion of β-cells than diabetic rats. Thus, ICV infusion of resistin sufficiently corrected glucose dysfunction by hyperplasia over a long period, which may account for glucose-stimulated insulin secretion as previously shown.

Leptin and insulin/IGF-1 signaling in hypothalamus
Because central leptin and resistin effects on glucose homeostasis exhibited similar patterns in normal and diabetic rats (with the effects slightly greater in diabetic rats), the hypothalamic signaling cascade was investigated in diabetic rats. ICV leptin infusion stimulated phosphorylation of STAT3Tyr705 in the hypothalamus without altering the STAT3 protein levels, whereas resistin rather decreased the phosphorylation (Fig. 5AGo). This suggests that hypothalamic leptin signaling was improved by leptin but decreased by resistin. In contrast to STAT3 phosphorylation, central leptin resulted in a significant and markedly decreased level of phosphorylation of AMPKThr172 without modifying the protein levels in the hypothalamus, whereas resistin did not decrease it. Leptin plus resistin infusion exhibited the summation of their alteration (Fig. 5AGo). When leptin was centrally infused, the phosphorylation of STAT3 and AMPK showed consistent results in improving energy sensing in the hypothalamus, leading to a reduction in food intake and body weight.


Figure 5
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  		FIG. 5. The modulation of insulin and leptin signaling in the hypothalamus at the end of experimental periods. The hypothalamus was collected from the rats at the end of experimental periods of ICV infusion of CSF, leptin, resistin, and leptin plus resistin (Lep+Res) and immediately lysed with a lysis buffer, and the phosphorylation and expression levels of the STAT3 and AMPK, involved in leptin signaling (A), were determined by immunoblotting (IB) with specific antibodies. In addition, tyrosine phosphorylation (py20) and expression of IRS2 was measured by immunoprecipitation (IP), followed by immunoblotting analysis (B). Serine phosphorylation and expression of Akt was determined by immunoblotting. The intensity of protein expression was determined using an Imagequant TL (Amersham Biosciences). These experiments were repeated four times for islets, and the values are mean ± SD. *, There were significant differences among the groups at P < 0.05; **, P < 0.01; a–d, values on the same parameter with different letters were significantly different at P < 0.05.

 
Leptin and resistin infusion did not modify IRS2 protein levels, indicating they were not associated with IRS2 induction (Fig. 5BGo). Similar to STAT3 phosphorylation, ICV infusion of leptin potentiated tyrosine phosphorylation of IRS2 in the hypothalamus, but resistin did not. Leptin infusion activated the phosphorylation of Akt in parallel with tyrosine phosphorylation of IRS2. Surprisingly, resistin infusion stimulated the phosphorylation of Akt even though tyrosine phosphorylation of IRS2 was not activated. Thus, the enhanced phosphorylation of Akt was not associated with increases or decreases in food intake in the resistin group, and resistin enhanced insulin signaling but not through IRS2.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Adipokines have been known to modulate feeding and energy expenditure through the central nervous system, where the hypothalamus plays a pivotal role in the transduction of peripheral afferents, which indicates satiety (12, 13). In addition, they regulate insulin resistance and glucose homeostasis, but the precise mechanism is unknown (25). Central leptin and resistin inhibit dopamine and norepinephrine release in the hypothalamic neuronal ending, where catecholamines can be regarded as central transducers of peripheral signaling (26). Furthermore, hypothalamic leptin delivers a central signal to the periphery through melanocortin pathways and sympathetic stimulation (15, 27). These results provide evidence that leptin and resistin work through central pathways, although their direct action in peripherals such as adipose tissues, liver, and β-cells cannot be ruled out (12, 13, 26). Taken together with recent studies on the metabolic actions of leptin and resistin (10, 27, 28), we hypothesize that central leptin and resistin modulate energy and glucose homeostasis via different pathways.

In agreement with other previous findings (10, 25), the present study exhibited an initial marked decrease of food intake among the rats, followed by a recovery during the later period when leptin-infused rats were fed a high-fat diet. Despite normalization in food intake, body weight in the rats infused with leptin remained at a lower level than the CSF control group throughout the experimental periods. This may be due to increased energy expenditure (27, 29). In the present study, ICV resistin appeared to lower food intake for the initial 3–4 d, but it did not modulate food intake during the rest of the experimental periods, whereas resistin offset the leptin effect on food intake. Similarly with food intake, ICV resistin did not alter body weight regardless of leptin infusion. Unlike body weight, epididymal fat pads were reduced more in the resistin and leptin plus resistin groups than in the control group and accompanied by reduced serum leptin levels. Tovar et al. (30) supported our findings that the central administration of resistin decreased transient and modest food intakes without affecting body weight in rats. Based on the results of the present study, we concluded that leptin and resistin act as potent modulators of the energy glucose homeostasis through the hypothalamus and that leptin and resistin regulates energy homeostasis through independent central pathways.

The mechanism by which the body weight is modulated by leptin and resistin is not clear. Leptin has been known to enhance energy homeostasis through potentiating STAT3 signaling in the hypothalamus (10, 12), and hypothalamic leptin and insulin signaling pathways interact through AMPK, an energy sensor and regulator of leptin’s action (31). ICV leptin has been reported to improve its STAT3 phosphorylation in the hypothalamus and attenuate its AMPK phosphorylation. However, very few studies have reported on the central effect of resistin on body weight. Our study showed that similar to leptin, central resistin affected insulin and leptin signaling pathways in the hypothalamus. ICV resistin decreased STAT3 phosphorylation in the hypothalamus, whereas resistin counteracted its promotion by leptin in central infusion of leptin plus resistin. However, AMPK phosphorylation was weakened by ICV resistin in comparison with CSF, even though the decrease was not as much as leptin. These results suggested that ICV resistin attenuated leptin signaling through STAT3 in the hypothalamus, which may lead to increased food intake. However, in the present study, resistin did not increase food intake compared with the control. This was explained by the attenuation of AMPK phosphorylation, resulting in a decrease of food intake. Thus, besides leptin signaling, another pathway may be involved in energy homeostasis to modulate the phosphorylation of AMPK.

Because AMPK acts as a mediator of energy homeostasis to integrate leptin and insulin signaling pathways (31, 32, 33), insulin signaling may be involved in resistin signaling in the hypothalamus. Hypothalamic insulin signaling is well known to regulate energy homeostasis through IRS2->phosphatidylinositol-3-kinase signaling and to interact with leptin and insulin signaling (34, 35). The present study showed that insulin signaling was potentiated with ICV resistin as well as leptin, which explained the attenuation of AMPK phosphorylation despite attenuated STAT3 phosphorylation in resistin-infused rats. These observations demonstrate that, in addition to the Janus kinase 2/STAT3 pathway, the phosphatidylinositol-3-kinase pathway is also required for energy homeostasis. These changes of the signaling pathways are transmitted into the peripheries through hormonal secretion and/or sympathetic nervous system to modulate the metabolic actions (15).

In addition to energy homeostasis, leptin and resistin were found to regulate glucose homeostasis in the liver and islets (8, 13). Some studies have been conducted to investigate the effect of central leptin on glucose homeostasis, exhibiting the enhancement of hepatic and muscle glucose regulation (15, 36). However, ICV leptin infusion, either in a single high dose or in more modest doses in mice, resulted in acute glucose intolerance, suggesting that leptin decreased insulin secretion through its actions in the central nervous system via melanocortin pathways (37, 38). In addition, several in vitro studies on islets and β-cells have shown that leptin decreased glucose-stimulated insulin secretion. Consistent with other studies of short-tem ICV leptin infusion (37, 38), we found that long-term ICV infusion of leptin decreased glucose-stimulated insulin secretion at hyperglycemic clamp. Thus, central and peripheral leptin attenuated β-cell function, which was overcome by the reduction of insulin resistance.

Unlike leptin, circulating levels of resistin are associated with elevated insulin resistance. The acute infusion of purified recombinant mouse resistin, designed to acutely elevate the levels of circulating resistin up to those observed in mice fed with a high-fat diet, elevated hepatic insulin resistance (6). Resistin is expressed in islets as well as adipose tissues and up-regulated in animals with elevated insulin resistance (39). Short-term treatment of 100 nM resistin elevated glucose-stimulated insulin secretion in isolated islets, whereas 1-d treatment failed to increase insulin release with a high glucose level, but it elevated insulin secretion with a low glucose level (40). Our preliminary study showed that short-term treatments of resistin elevated glucose-stimulated insulin secretion up to 50 nM in a dose-dependent manner, but higher dosages of resistin rather initiated a decrease in secretion (data not shown). This indicated that higher dosage or long-term resistin treatment impaired β-cell function by disrupting their regulation. Elevated circulating levels of resistin exhibited harmful effects on glucose homeostasis in the liver and islets. However, the effects of central resistin on peripheral glucose homeostasis have recently begun to be studied. The acute hypothalamic infusion of resistin increased hepatic insulin resistance by glycogenolysis, but it did not alter gluconeogenesis, and there were no changes in phosphoenolpyruvate carboxykinase and glucose-6-phosphatase expression (41). However, in our study, long-term ICV infusion of resistin improved hepatic insulin sensitivity by improving β-cell function and mass in comparison with the control group. This may be associated with the improvement of hepatic insulin sensitivity and insulin secretion capacity directly through the central pathway or indirectly through the reduction of serum resistin levels.

In conclusion, leptin and resistin may regulate peripheral energy and glucose homeostasis through a central effect on the hypothalamus in normal and diabetic rats, and this effect is mediated through leptin and insulin signaling. This study further supports the notion that central leptin and resistin modulate peripheral glucose homeostasis in independent pathways; leptin strongly improves insulin sensitivity but decreases insulin secretion by potentiating hypothalamic leptin and insulin signaling by stimulating IRS2 phosphorylation, whereas resistin moderately enhances insulin sensitivity and strongly improves glucose-stimulated insulin secretion by stimulating Akt phosphorylation without altering IRS2 phosphorylation. Additional studies are needed to investigate the modulation of hepatic insulin signaling and inflammatory signaling through central leptin and resistin.


   Footnotes
 
This work was supported by a grant from the Korean Science and Engineering Foundation in Korea (R01-2006-000-10389-0).

Disclosure Statement: The authors have nothing to disclose.

First Published Online November 8, 2007

See editorial p. 443.

Abbreviations: AMPK, AMP kinase; BrdU, 5-bromo-2-deoxyuridine; CSF, cerebrospinal fluid; En%, energy percent; GIR, glucose infusion rate; ICV, intracerebroventricular; IRS, insulin receptor substrate; OGTT, oral glucose tolerance test; Px, pancreatectomy; STAT3, signal transducer and activator of transcription 3.

Received June 8, 2007.

Accepted for publication October 31, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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