Cooperative effects of galanin and leptin on alleviation of insulin resistance in adipose tissue of diabetic rats

Abstract It was reported that either orexigenic neuropeptide galanin or anorexigenic hormone leptin caught benefit insulin sensitivity through increasing the translocation of glucose transporter 4 (GLUT4) in patients with diabetes. To date, it is unknown whether galanin can potentiate the effect of leptin on alleviation of insulin resistance. Therefore, in the current study we sought to assess the combined effect of central leptin and galanin on insulin resistance in the adipose tissues of type 2 diabetic rats. Galanin and leptin were injected into the intracerebroventricle of the diabetic rats, respectively, or cooperatively once a day for 2 weeks. Then, several indexes of insulin resistance were examined. The results showed that glucose infusion rates in the hyperinsulinaemic‐euglycaemic clamp test, plasma adiponectin content and GLUT4 translocation, as well as Akt phosphorylation in fat cells, were higher, not GLUT4 protein and GLUT4 mRNA expression, but HOMA index was lower in the galanin + leptin group than either one of them. Furthermore, treatment with MK‐2206, an Akt inhibitor, blocked the combined effects of galanin + leptin on alleviation of insulin resistance. These results suggest that galanin can improve the leptin‐induced mitigative effects on insulin resistance in the fat cells, and those provided new insights into the potential tactics for prevention and remedy of insulin resistance.


| INTRODUC TI ON
The hallmark of insulin resistance is a disturbance in glucose uptake, resulting in hyperinsulinaemia and heperglycaemia. The relation between insulin resistance and subsequent risk of type 2 diabetes is well established. Despite extensive investigations into insulin resistance, the precise mechanism is incompletely understood. A lot of endocrine hormones, including galanin and leptin, are involved in the pathogenesis of insulin resistance for their multiple effects on glucose and lipid metabolism.
Data indicate that galanin can regulate energy homeostasis and insulin sensitivity of animals. Galanin neurons and its three subtype receptors 1-3 distribute throughout the brain, particularly in the dorsomedial hypothalamus, 1 where is an important regulative centre of energy metabolism. 2 An injection of galanin into paraventricular nucleus significantly increased feed intake and bodyweight, as well as circulating non-esterified fatty acid and lipoprotein lipase levels of the rats. 3 Animals with metabolic disorder of galanin are easy to develop type 2 diabetes mellitus. 4 Compared with controls, the galanin knockout mice reduced insulin sensitivity and insulin-independent glucose elimination, and their food intake, 5,6 whereas the homozygous galanin transgenic mice exhibited increased metabolic rate of lipid and carbohydrate for improved insulin sensitivity. 7 The results of our 8 and other's studies [9][10][11][12] indicated that an intracerebroventricular or abdominal administration of M35, a galanin antagonist, enhanced plasma glucose levels but reduced insulin sensitivity in diabetic rats.
Leptin is a protein synthesized and secreted by adipocytes.
Plasma leptin levels in humans and animals are correlated to body fat depot. 13 Leptin receptors are expressed in the arcuate and ventromedial regions of the hypothalamus, and adipose tissues. 14,15 Central administration of leptin suppressed bodyweight via decreasing food intake and stimulating energy expenditure of rats. 16 The leptin transgenic animals showed amelioration of high-fat-dietinduced insulin resistance, 17,18 whereas the leptin-deficient ob/ob mice displayed obesity and insulin resistance with hyperinsulinaemia and hyperglycaemia, 19 which might be reversed by exogenous leptin. 20 Intriguingly, LepRb, a long-isoform leptin receptor, is expressed in galanin neurons of brainstem and hypothalamus of mice. 21 The double-labelling studies indicated that co-localization of galanin and leptin receptors distributed in the periventricular nucleus, the arcuate nucleus, the supraoptic nucleus and the lateral hypothalamus. 15 This co-localization of galanin and leptin receptors offers an morphologic basis of their interaction. The galanin-expressing neurons in the periventricular nucleus are a target of leptin to regulate feeding behaviour and energy expenditure, 15,22 and galanin is an important mediator of leptin action to modulate nutrient reward. 23 Deletion of galanin gene amplifies leptin-induced weight loss, suggesting that there is an interaction between galanin and leptin signalling system. 21 However, the combined effects of central leptin and galanin on insulin resistance have not been identified. Therefore, the current experiment was designed to evaluate the combined anti-diabetic effects of both hormones in fat cells of type 2 diabetic rats.

| Animal preparation
The experiment was performed on 120 freely moving male Wistar rats weighing 150 ± 10 g. All animals were housed in group on a 12:12-h light-dark cycle with ad libitum access to high-fat food (59% fat, 21% protein and 20% carbohydrate) and water in a climate-controlled environment (22 ± 2°C at 50%-60% humidity) for 8 weeks. Then, the rats were treated with 30 mg/kg streptozotocin intraperitoneally. 11 After another 4 weeks, the animals with over 11.1 mmol/L fasting blood glucose levels were used as diabetic models. All experiments were conducted according to the Guiding Principles for Care and Use of Experimental Animals to minimize the animal suffering, and the experiment was approved by the Tongji University Ethics Committee.

| Animal groups
A total of 112 model rats were randomly divided into seven groups of 16 each: diabetic control, galanin group, leptin group, galanin + leptin group, galanin + MK-2206 group, leptin + MK-2206 group and both hormones + MK-2206 group. Besides, 16 rats with normal glucose levels were included in the healthy control group.

| Intracerebroventricular injection
All rats were anaesthetized with 50 mg/kg amobarbital sodium (i.p.) and implanted stereotaxically with a guide cannula into the lateral ventricle: anterior-posterior (AP), −0.8 mm; L, 1.4 mm; and V, 3.3 mm. 11 The cannula was fixed to the skull by stainless steel screws and dental cement. After recovered from the surgery for 7 d, rats in six agent-treated groups received an i.c.v. infusion of leptin (0.6 nmol), galanin (0.1 nmol) and MK-2206 (300 μg/kg), either, respectively, or in combination, once a day for continuous 14 days.
Both control groups were intraventricular infusion with the same volume of artificial cerebrospinal fluid.

| Hyperinsulinaemic-euglycaemic clamp and 3 H-2DG experiments
Fasted for 12 hours after the last injection, half of the rats in every group (n = 8) were anaesthetized as above and cannulated in the jugular vein for infusion of glucose and insulin and in the carotid artery for sampling. 12 Insulin was constantly infused in 2 mU/kg·min velocity, and 10% glucose was instilled at variable rates as needed to keep glucose levels at 5 ± 0.5 mmol/L. The glucose injection rate was calculated according to six samples under homeostasis during the experiment.
After fasted for 12 hours, the remaining rats in every group (n = 8) were anaesthetized as above and received an intraperitoneal injection with 250 mg/kg 3 H-2DG. At 30 minutes after the treatment, epididymal fat pad and 4 mL artery blood were fast collected and stored at -80°C.

| Subcellular fractionation
The washed and minced fat pads were homogenized with homogenization buffer at 4°C. 10 Then, the homogenate was centrifuged at 13 000 g for 20 minutes at 4°C. The 3 H-2DG uptake was calculated with part of the supernatant in a liquid scintillation counting (Tri-Carb 2000, Packard Instrument Co.). The intracellular membranes were obtained by recentrifugation of remaining supernatant at 31 000 g for 60 minutes. The pellet after this spin was layered over a sucrose cushion and centrifuged at 75 000 g for 60 minutes. The pellet was re-spun at 39 000 g for 20 minutes to yield plasma membranes.

| HOMA index
The homeostasis model assessment (HOMA)-insulin resistance indexes were measured by insulin levels (mU/mL) × blood glucose levels (mmol/L)/22.5. The blood insulin and glucose levels were measured using competitive insulin ELISA kits and glucometer (HMD Biomedical, Taiwan), respectively. All measurements were performed in duplicate, and the mean of two measurements was considered.

| Measurement of plasma adiponectin levels
The fast blood adiponectin levels were quantified using competitive ELISA kits in accordance with the manufacturer's directions.

| Total RNA extraction and real-time PCR
Total RNA was prepared using TRIzol reagent from the adipose tissue. 10 After determination of RNA concentrations by measuring the absorbance at 260-280 nm, 4 μL RNA as template was re-

| Western blotting
The 50 mg protein samples from the subcellular fractions were separated with a 12% SDS-PAGE and transferred to a nitrocellulose membrane. 11 The membranes were then probed with a primary antibody against GLUT4, Akt and pAkt, respectively, and a HRP-conjugated secondary antibody successively. The signals were detected with chemiluminescence and quantified by densitometry with a HPIAS-2000 Image Analysis System.

| Statistical analysis
All data were expressed as mean ± SEM. Differences between groups were determined via the two-way analysis of variance, followed by the Tukey's test. A P value <.05 was statistically considered significant.
In addition, bodyweight and food intake reduced by 12.3% (P < .05) and 13.6% (P < .05) in the galanin + leptin+MK-2206 group (DGLM) compared with the galanin + leptin group (DGL), by 14.4% (P < .01) and 17.2% (P < .01) in the galanin + MK-2206 group (DGM) compared with DG, and by 12.1% (P < .05) and 14.5% (P < .05) in the leptin group (DL) compared with the diabetic controls (DC). However, bodyweight and food intake increased by 13.8% (P < .05) and 14.9% (P < .05) in DGL F I G U R E 1 The central effect of galanin and leptin on bodyweight and food intake of rats (n = 8). The bodyweight and food intake of animals were lower in the galanin + leptin group (DGL) or the galanin + MK-2206 group (DGM) than the galanin group (DG), the both hormones + MK-2206 group (DGLM) than DGL, and the leptin group (DL) than diabetic controls (DC). But both indexes were higher in DGL or the galanin + MK-2206 group (DLM) than in DL, in DG than DC. The bodyweight was lower and food intake was higher in DC than the healthy controls (HC). Difference in the both indexes was non-significant between DGLM and DGM, and between DGLM and DLM. The data are shown as the means ± SEM. ○P < .05, ○○P < .01 vs HC; •P < .05 vs DC; △P < .05, △△P < .01 vs DG; □P < .05 vs DL; *P < .05 vs DGL compared with DL, by 12.9% (P < .05) and 14.9% (P < .05) in the leptin + MK-2206 group (DLM) compared with DL, and by 10.9% (P < .05) and 13.4% (P < .05) in DG compared with DC. The bodyweight was lower, and the food intake was higher in DC than in the healthy controls (HC). Differences in the both indexes were non-significant between DGLM and DGM, and between DGLM and DLM (P > .05).

| D ISCUSS I ON
The purpose of current study was to investigate the combined ef- and insulin sensitivity. 26 The second is the glucose infusion rates in hyperinsulinaemic-euglycaemic clamp tests, which is a classical measurement of insulin sensitivity. 12 The third is GLUT4 translocation from intracellular storage organelles onto plasma membranes.
It is acknowledged that only after transported onto the cell surface, GLUT4 can move glucose into cells. So, the GLUT4 levels in plasma membranes of cells reflect glucose clearance and insulin sensitivity. 9,27 The fourth is the plasma adiponectin concentration.
Secreted by adipose tissues, adiponectin shows anti-inflammatory and anti-diabetic properties. 28 Adiponectin-deficient mice exhibited insulin resistance and diabetic symptom. 29  Noteworthily, the GLUT4 mRNA level more likely reflects the change in GLUT4 synthesis rate rather than its half-life. 11 In this experiment, GLUT4 mRNA and total GLUT4 protein expression levels were almost changeless in the galanin + leptin group compared with leptin-or galanin-treated group, suggesting that co-administration of both hormones was unable further to enhance the GLUT4 levels in comparison with anyone did in the fat cells.
There is an interaction between leptin and galanin in their synthesis and secretion. Treatment with galanin reduced leptin expression and secretion in a dose-dependent manner in the adipose tissue of fasting rats. 31 Accordingly, galanin knockout mice showed increased circulating leptin levels and leptin sensitivity to reduce their bodyweight and fat pad mass, 32 that is deletion of galanin gene amplified leptin-induced weight loss. 33 In turn, leptin significantly increased the galanin serum level and gene expression in the hypothalamus. 33,34 Thus, there is a negative feedback in their synthesis between leptin and galanin, the former to the latter is promoting, and the latter to the former is inhibitive. This negative feedback is helpful to maintain the homeosta of both hormones to keep energy homeostasis.
In order to understand the mechanism of cooperation between galanin and leptin in amelioration of insulin resistance, we surveyed Akt phosphorylation, a key link of signalling systems of both hormones. 35,36 Recent studies showed that leptin receptor mediated the regulation of the glucose metabolism via activation of AKT pathway. 37 The AKT inhibitor can significantly counteracted the effect of leptin, and AKT activator can counteracted the effect of leptin silencing. In addition, galanin can benefit glucose uptake via inhibition of adenylyl cyclase and cAMP through Gi/o receptors to activate the PI3K/Akt signalling pathway too. 38 Thus, Akt becomes a meeting point of both signalling pathways to regulate glucose uptake and metabolism. This overlap may result in enhancing the signal intensity and transmitting efficacy to increase insulin sensitivity. Yuan et al found that galanin might modulate the leptin signals, and the inhibiting effects on neuron activity were stronger after co-application of galanin and leptin into the gastric compartment than application of galanin or leptin alone in the nucleus tractus solitarius. 39 In the current study, combination of both hormones stimulated more Akt phosphorylation than either leptin or galanin alone did, which may be blocked by MK-2206, suggesting that Akt was necessary for the cooperative effects of both hormones on alleviation of insulin resistance.
In summary, the results of our present study showed that the central effect of galanin and leptin on appetite and bodyweight of animals is opposite, but that on amelioration of insulin resistance is cooperative. The co-administration of both hormones further enhanced glucose infusion rates in the clamp tests, plasma adiponectin content, 2-deoxy-D-[ 3 H]glucose uptake, GLUT4 translocation and Akt phosphorylation in the adipose tissues, but reduced HOMA index, did not affect GLUT4 mRNA and total GLUT4 expression levels. Besides, the Akt phosphorylation is necessary for the cooperative effects of both hormones in the fat cells. These findings deepen our understanding of the interrelation between galanin and leptin in mitigation of insulin resistance, and provide an experimental clue for further testing whether co-administration of both hormones may get better efficacy against insulin resistance than treatment with galanin or leptin alone for type 2 diabetic patients.

ACK N OWLED G EM ENTS
We would like to thank Dr Xusheng Chang for collection of part experimental data. This work was supported by the Grant of National Natural Scientific Fund of China (81070238 and 81001572).

CO N FLI C T O F I NTE R E S T
The authors have no conflicts of interest to disclose.

AUTH O R CO NTR I B UTI O N S
LB and SQ contributed to the design and writing of the manuscript.
L.B and XC were involved in conduct/data collection and critical analysis.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.