Two human PC cell lines, BxPC3 and Panc-1, and normal pancreas cells HPC-Y5 were purchased from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. The cells were cultured in a 37°C, 5% CO₂ incubator (Thermo Forma 3111; Thermo Fisher Scientific, Inc.) in RPMI-1640 medium (cat. no. SH30809.01B; HyClone; GE Healthcare Life Sciences), which contained 10% FBS (cat. no. 16000-044; Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin (cat. no. P1400-100; Beijing Solarbio Science & Technology Co., Ltd.). The medium was refreshed every two days during incubation.

Short hairpin RNA (shRNA)-1 and shRNA-2 targeting two different sites of LepR (GenBank no. BC131779.1; Table I) were constructed and inserted into the Agel I/Ecol I restriction sites of a pLKO.1-puro vector (Addgene, Inc.). Following confirmation by DNA sequencing (Shanghai Majorbio Pharmaceutical Technology Co., Ltd.), LepR-shRNA-1 or LepR-shRNA-2 was co-transfected with the viral packaging plasmids psPAX2 and pMD2G (Addgene, Inc.) into 293T (The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences) cells using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) in a 37°C incubator. The virus particles were collected by ultracentrifugation 48 h post-transfection.

BxPC3 cells were transduced with lentiviruses expressing shRNA negative control (shNC), LepR-shRNA-1 and LepR-shRNA-2; medium-treated cells were used as the untransfected control. Following 48 h of transduction by lentivirus infection in a 37°C, 5% CO₂ incubator, the silencing efficiency of LepR-shRNA-1 and LepR-shRNA-2 were evaluated by reverse transcription-quantitative PCR (RT-qPCR) and western blot analysis. Subsequently, cell proliferation, glucose uptake and lactate production, as well as the expression of several related genes and proteins including LepR-short, LepR-long, HKII, GLUT1, AKT and p-AKT, were measured at 48 h of transduction.

BxPC3, Panc1 or HPC-Y5 cells were treated with 0, 20, 50 or 100 ng/ml Leptin. Subsequently, cell proliferation, glucose uptake, lactate production as well as the expression of HKII and GLUT1 were detected. The treatment groups for BxPC3 cells were: i) RPMI-1640 medium (control); ii) shRNA negative control (shNC); iii) LepR-shRNA-1 (shRNA-1); iv) LepR-shRNA-2 (shRNA-2), or i) Medium + DMSO (control); ii) 50 ng/ml leptin + DMSO; iii) medium + 25 µmol/l LY294002 (AKT inhibitor); and iv) 50 ng/ml leptin + 25 µmol/l LY294002. A subset of shNC and shRNA-2-transfected BxPC3 cells were further treated with DMSO or 50 ng/ml IGF-1 (AKT activator). Following treatment, cell proliferation, glucose uptake, lactate production, and the expression of several related genes (HKII, GLUT1, AKT and p-AKT) were examined.

The proliferation of treated human PC cells (BxPC3 and Panc1) and normal pancreas cells HPC-Y5 were evaluated by the Cell Counting Kit-8 assay (CCK-8; cat. no. CP002; SAB Biotherapeutics, Inc.). Human PC cells were seeded in 96-well plates (3×10³ cells/well) and cultured in a 37°C, 5% CO₂ incubator overnight. Following treatment for 24 h, 100 µl CCK-8 solution (diluted 1:10 in serum-free medium) was added and incubated for 1 h in a 37°C, 5% CO₂ incubator. Subsequently, the absorbance value [optical density (OD)] at 450 nm was determined using a microplate reader (DNM-9602; Perlong Medical Equipment Co., Ltd.).

Human PC cells (BxPC3 and Panc1) and normal pancreas cell HPC-Y5 in the logarithmic growth phase were inoculated in 6-well plates (5×10⁵ cells/well) and cultured in a 37°C incubator overnight. After treatment with a range of leptin concentrations (0, 20, 50 and 100 ng/ml) or according to the grouping aforementioned, the cells were cultured for 3 h in low-glucose DMEM, followed by washing with glucose-free Krebs-Ringer bicarbonate buffer (containing 2% BSA) at 37°C. The cells were subsequently incubated in glucose-free DMEM containing 100 µM 2-NBDG (cat. no. 0467597-16; Cayman Chemical Company) for 45 min, and glucose uptake was evaluated using a 2-NBDG Glucose Uptake Assay kit (Nanjing Jiancheng Bioengineering Institute). Lactate production was evaluated using a Lactate Assay Kit (Nanjing Jiancheng Bioengineering Institute). The supernatant of the treated cells was prepared according to the manufacturer's protocol and the OD was measured at 530 nm using a spectrophotometer.

Following treatment, total RNA from human PC cells (BxPC3 and Panc1) and normal pancreas cells HPC-Y5 was extracted using TRIzol reagent (cat. no. 1596-026; Invitrogen; Thermo Fisher Scientific, Inc.). Following RNA quantification and confirmation of RNA integrity by electrophoresis with 1% gel, 1 µg of RNA was reverse transcribed into cDNA using the RevertAid First strand cDNA synthesis kit (cat. no. K1622; Fermentas; Thermo Fisher Scientific, Inc.). RT-qPCR was performed in triplicate using an ABI-7300 Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific, Inc., USA) and the Maxima SYBR Green/ROX qPCR Master Mix kit (cat. no. K0223; Thermo Fisher Scientific, Inc.). GAPDH was used as an internal reference. mRNA expression levels of HKII, GLUT1, LepR-common, LepR-short and LepR-long were analyzed using the 2^−ΔΔCT method (28). The primers used were as follows: HKII, forward 5′-ACGACAGCATCATTGTTAAGG-3′, reverse 5′-TTTGGCAAAGTGAGGATGTAG-3′; GLUT1, forward 5′-TGCAGGAGATGAAGGAAG-3′, reverse 5′-CAATGGTGGCATACACAG-3′; LepR-common, forward 5′-TTGTGCCAGTAATTATTTCCTCTT-3′, reverse 5′-CACACCAAAGAATGAAAAAGCTAT-3′; LepR-short, forward 5′-TTCCTGGGCACAAGGACTTA-3′- reverse 5′-GCTCCAAAAGAAGAGGACCA-3′; LepR-long, forward 5′-TTCCTGGGCACAAGGACTTA-3′, reverse 5′-TTTGTGTCCCTGGGTACTTGA-3′; GAPDH, forward 5′-AATCCCATCACCATCTTC-3′, reverse 5′-AGGCTGTTGTCATACTTC-3′. The thermocycling conditions were as follows: 95°C for 10 min; followed by 40 cycles of 95°C for 15 sec and 60°C for 45 sec (29).

Following the various treatments, total protein from human PC cells (BxPC3 and Panc1) and normal pancreas cells HPC-Y5 were extracted using RIPA buffer supplemented with protease and phosphatase inhibitors (cat. no. R0010; Beijing Solarbio Science & Technology Co., Ltd.), followed by quantification by the BCA Kit (cat. no. PICPI23223; Thermo Fisher Scientific, Inc.). Proteins (25 µg) were separated using 10 (spacer) and 8% (separation) SDS-PAGE, and transferred to PVDF membranes (cat. no. HATF00010; EMD Millipore). Subsequently, the membranes were blocked in 5% skimmed milk (cat. no. BYL40422; BD Biosciences) for 1 h at room temperature and incubated with primary antibodies against GLUT1 (1:1,000; cat. no. ab115730; Abcam), HKII [1:1,000; cat. no. 2867; Cell Signaling Technology (CST)], AKT (1:1,000; cat. no. 2920; CST), phosphorylated (p)-AKT (1:2,000; cat. no. 4060; CST), LepR-long (1:250; cat. no. sc-1835; Santa Cruz Biotechnology, Inc., California, USA), LepR-short (1:250; catalog no. sc-8325; Santa Cruz Biotechnology, Inc.) and GAPDH (1:1,000; cat. no. 5174; CST) overnight at 4°C with gentle agitation. Following 5–6 washes in TBS + 0.1% Tween-20, the membranes were incubated for 2 h at room temperature with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:1,000; cat. no. ZB2301, OriGene Technologies, Inc.). The blots were visualized by ECL reagent (cat. no. WBKLS0100; EMD Millipore) and images were captured with a Tanon-5200 ECL imaging system (Tanon Science and Technology Co., Ltd.). Protein expressions levels were normalized to GAPDH and analyzed using ImageJ 1.47v (National Institutes of Health).

All statistical analyses and calculations in this study were carried out using GraphPad Prism 7.0 software (GraphPad Software, Inc.). The statistical significance of differences between the two groups was determined using two-tailed Student's t-test, and multiple comparisons were made by one-way ANOVA followed by Tukey's test for multiple comparisons. All experiments were performed in triplicate, and data are expressed as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.

Human PC cell lines, BxPC3 and Panc1, and normal pancreas cells were treated with increasing concentrations of leptin (0, 20, 50 and 100 ng/ml) for 24 h. In vitro treatment with leptin significantly stimulated the proliferation of BxPC3 and Panc1 cells as well as pancreas cells HPC-Y5 in a dose-dependent manner (Fig. 1A). These results indicated that leptin stimulation may promote the proliferation of human PC and pancreas cells. In addition, glucose uptake (Fig. 1B) and lactate production (Fig. 1C) of BxPC3, Panc1 and pancreas cells were markedly increased by leptin stimulation, accompanied by increased expression of HKII and GLUT1 (Fig. 1D and E). These data suggested that leptin stimulation may contribute to glucose metabolism and proliferation of human PC cells and healthy pancreatic cells.

Due to the significant increase of proliferation, glucose uptake and lactate production in both BxPC3 and Panc1 cells, in the present study, one cell line, BxPC3, was selected for further investigation. BxPC3 cells were infected with lentiviruses expressing shNC, LepR-shRNA-1 and LepR-shRNA-2; untreated cells were used as a control. mRNA expression of LepR-common, LepR-short and LepR-long (Fig. 2A), as well as the protein expression levels of LepR-short and LepR-long, were significantly downregulated by LepR-shRNA-1 and LepR-shRNA-2 (Fig. 2B). Therefore, the LepR-shRNA-1 and LepR-shRNA-2 vectors were used for subsequent experiments.

To investigate the role of leptin in human PC cells, proliferation, glucose uptake and lactate production were assessed in BxPC3 cells following LepR silencing. The results indicated that the proliferation of BxPC3 cells was notably suppressed by silencing of LepR, compared with the control groups (Fig. 3A). Glucose uptake (Fig. 3B) and lactate production (Fig. 3C) of BxPC3 cells were also inhibited, accompanied by decreased protein expression levels of HKII, GLUT1 and p-AKT, whereas total AKT protein expression was unaltered (Fig. 3D), suggesting an inhibitory effect of leptin silencing on AKT activation. These results demonstrated the beneficial effects of leptin in glucose metabolism and proliferation of human PC cells, which may be involved in AKT pathway activation.

The molecular mechanisms of leptin in mediating glucose metabolism and proliferation of human PC cells were explored. Leptin-stimulated proliferation, glucose uptake and lactate production were effectively counteracted by co-treatment with the AKT inhibitor LY294002 (Fig. 4A-C, respectively). The increased protein expression levels of HKII, GLUT1 and p-AKT induced by leptin stimulation were also significantly decreased (Fig. 4D), whereas total AKT was unchanged (Fig. 4D). Leptin stimulation potently attenuated the effects of AKT inhibitor, LY294002, suggesting that there was a rescue effect of leptin stimulation on AKT inhibition. In addition, the inhibition of proliferation, glucose uptake and lactate production induced by Lep2-shRNA-2 transfection were significantly counteracted by co-treatment with the AKT activator IGF-1 (Fig. 4E-G, respectively), accompanied by an increased expression of GLUT1 and HKII protein (Fig. 4H). In the present study, Lep2-shRNA-2 was used due to its better downregulation efficiency compared with shRNA-1. These results further demonstrated that leptin may stimulate glucose metabolism and proliferation of human PC cells potentially through activating the AKT pathway.