Methods
Animals
Thirty-six adult male Wistar rats (250 ± 10 g) were caged with a 12-h light/dark cycle and given standard chow and water
ad libitum. After an overnight fast, rats were anesthetized and positioned in a stereotaxic apparatus. A cannula attached to an osmotic minipump (Alzet, Durect Corporation, Cupertino, CA) containing saline (controls, C) or leptin (Preprotech, Rocky Hill, NJ, USA; 12 μg/day) was implanted and maintained during 14 days (L), as reported [
6]. To discriminate the inhibitory effect of leptin on food intake, a pair-fed group (PF) was included. On the last day, twelve rats were fasted for 12 h and then sacrificed, obtaining trunk blood for the determination of glucose, leptin and insulin levels. The liver was weighed and processed for measurement of activation of insulin signaling targets, protein levels of glucose transporter (GLUT)2 and −4 and phosphoenolpyruvate carboxykinase (PEPCK). The weight of the gastrocnemius and subcutaneous and epididymal fat pads was also recorded.
Twelve rats were fasted for 12 h, followed by an oral glucose tolerance test (OGTT) (n = 4 per group). A bolus of glucose (2 g/kg body weight) was administered orally [
9]. Glycemia was determined (Accu-Check Sensor) in blood samples extracted from the tail vein before glucose administration and at 15, 30, 60 and 120 min, as well as insulin levels. The liver was processed after OGTT for measurement of free glucose and glycogen concentrations.
Insulin sensitivity was assessed after fasting by performing an intraperitoneal insulin tolerance test (IPITT) [
10] in the remaining twelve rats. After the injection of 1 U/kg of insulin, blood samples were drawn at 30, 60, 90 and 120 min for glucose measurements. The liver was extracted for determination of glucose and glycogen. This study was approved by the Ethics Committee of the Universidad de Alcalá de Henares.
ELISAs
Serum leptin and insulin levels were measured using ELISA kits from Millipore Corporate Headquarters (Billerica, MA, USA). The intra- and inter-assay variations were lower than 10 %.
Western blotting
Western blots were performed using antibodies against GLUT2, the beta chain of the insulin receptor (IRβ) and PEPCK from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and anti-GLUT4 from Millipore (Temecula, CA, USA). The proteins were detected by chemiluminiscence using an ECL system. Quantification of the bands was carried-out by densitometry using a Kodak Gel Logic 1500 Image Analysis system and Molecular Imaging software 4.0 (Rochester, NY, USA). Proteins were normalized with β-actin (Thermo Scientific, Fremont, CA, USA).
Multiplexed bead immunoassay
Phosphorylated and total protein levels of IR substrate 1 (IRS1), Akt and phosphatase and tensin homolog on chromosome 10 (PTEN) were determined by a multiplexed bead immunoassay (Millipore). A minimum of 50 beads per parameter were analyzed in the Bio-Plex suspension array system 200 (Bio-Rad). Raw data (median fluorescence intensity, MFI) were analyzed with the Bio-Plex Manager Software 4.1 (Bio-Rad Laboratories).
Measurement of hepatic glucose and glycogen
Glucose was measured by an enzymatic method from Sigma-Aldrich (GAGO-20), in homogenized samples [
11]. For quantification of glycogen, liver samples were processed as previously reported [
6] and the resulting glucose concentrations determined by the same method.
Statistical analysis
Data are expressed as mean ± SEM. Statistical analysis was carried out by one-way ANOVA or repeated measures for OGTT or IPITT followed by a Bonferroni’s test. Values were considered significantly different when the P value was less than 0.05. Analyses were conducted with Prisma software 4.00 (GraphPad, San Diego, CA, USA).
Discussion
The goal of this study was to examine the effect of central leptin infusion on glycemia after a peripheral increase in glucose or insulin and the possible relationship with changes in glucose uptake and its metabolism in the liver. We found that leptin-treated rats had higher hepatic glucose and glycogen concentrations, probably due to higher levels of GLUT2 [
12], thus regulating glycemia. Several differences between pair-fed and leptin-treated rats were observed, in particular, higher leptin concentrations in the leptin- infused group. In fact, leptin infusion causes hyperleptinemia [
13] as intracerebroventricular leptin goes to the periphery, as previously reported [
14]. In addition, the gastrocnemius of these rats weighs more than in the pair-fed group, as previously reported [
15], probably related to the leptin-induced increase in carbohydrate disposal [
16]. Likewise, the reduction in fat pads is most likely due to leptin’s suppression of glucose utilization [
17].
Consistent with these findings, central leptin administration modifies glucose fluxes and production [
18,
19] and these changes are partially mediated by increasing hepatic insulin sensitivity, as we report here. In fact, exogenous leptin has been shown to exert positive effects on peripheral insulin signaling that involve leptin-insulin cross-talk [
1]. Indeed, an increase in central leptin is reported to reverse hepatic insulin resistance [
7] and to correct peripheral glucose usage [
20]. The insulin and leptin signaling pathways share several targets, such as Janus kinase-2, IRSs and Akt [
21], and we have reported that interaction of these pathways potentiates insulin signaling [
6]. While muscle most likely participates in the regulation of serum glucose levels, as leptin increases insulin sensitivity in this tissue [
22], our results clearly indicate a key role of the liver in leptin’s effects on serum glucose improvement.
Tolerance tests give more accurate information than homeostasis model assessment of insulin resistance to determine insulin sensitivity [
23,
24]. Here, tolerance tests reveal that the higher concentrations of glucose and glycogen in the liver of leptin-infused rats may be related with its increased insulin sensitivity. These changes seem to be due to the higher degree of phosphorylation on both the Thr308 and Ser473 residues of Akt, which is necessary to achieve full activation of the insulin signaling cascade [
25].
In conclusion, our results suggest that improvement in glycemia after peripheral glucose or insulin administration in central leptin-infused rats is due, at least in part, to the previous activation of hepatic insulin signaling that may increase glucose uptake and glycogen storage, thus contributing to lower serum glucose levels.
Acknowledgements
Authors thank Francisca Díaz for excellent technical assistance. This work was supported by the Spanish Ministry of Science and Innovation with the help of European FEDER funding (FIS PI13/02195), Ministerio de Ciencia e Innovación (BFU2011-27492 and BFU2014-51836-C2-2-R), the Network Center for Biomedical Research on Obesity and Nutrition (CIBEROBN) Instituto Carlos III and Fundación Endocrinología y Nutrición. S.C. is supported by CIBEROBN.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
EBR, SC and EAF performed animal treatments and experiments. LMF and JAC contributed to analysis and interpretation of data. EBR, JA and VB contributed to interpretation and discussion of the results and VB formulated the hypothesis and wrote the manuscript. All authors critically revised the article and approved the final version.