The tissues were processed for qRT-PCR analysis as previously described28, 29. Briefly, 48 h after adenovirus injection, rats were fasted for 24 h. After that, rats were anesthetized with isoflurane and i.c.v. injected with 10 μL of saline buffer (128 mM NaCl, 3 mM KCl, 1.3 mM CaCl2, 1.0 mM MgCl2, 1.3 mM NaH2PO4, 21 mM Na2HPO4, pH 7.4 and 320 mOsm) or 10 μL of 50 mM D-glucose diluted in the same buffer (320 mOsm, pH 7.4) at a rate of 2.5 μL/min. Hypothalamic samples were collected 2 h post-glucose or saline injection for the mRNA analysis expression. qRT-PCR analysis was used to measure the expression of hypothalamic cyclophilin (the housekeeping gene), GK, NPY, CART, POMC and AgRP. After the experimental treatments, the brain of each rat (six per condition) was removed, and the hypothalamic area was isolated and further dissected to obtain a region close to the 3V ependymal layer. All microdissections were performed closest to the basal 3V and under a stereomicroscope Leica M80 (Leica, Wetzlar, Germany). Total RNA from hypothalamus was isolated using TRIzol (Invitrogen) and treated with DNase I (Fermentas International INC, Burlington, Ontario, Canada) to remove genomic DNA contamination. A total of 2 µg of RNA from each sample was reverse transcribed into cDNA according to the manufacturer’s protocol of M-MULV reverse transcriptase (Fermentas International INC). Parallel reactions were performed in the absence of reverse transcriptase to control for the presence of genomic DNA. qRT-PCR reactions were prepared with a Brilliant II SYBR Green QPCR Master Mix kit (Agilent Technologies, Inc., Santa Clara, CA, USA) in a final volume of 12.5 µL containing 1x SYBR green Master Mix, 1 µL cDNA sample and 500 nM of the following sets of primers: cyclophilin, sense 5′-ATA ATG GCA CTG GTG GCA AGT C-3′ and antisense 5′ATT CCT GGA CCC AAA ACG CTC C3′ (expected product of 239 bp); GK, sense 5′AAA GAT GTT GCC CAC CTA CGT GCG3′ and antisense 5′ATC ATG CCG ACC TCA CAT TGG C3′ (expected product of 510 bp); NPY, sense 5′-TGT TTG GGC ATT CTG GCT GAG G-3′ and antisense 5′- CTG GGG GCA TTT TCT GTG CTT TC-3′ (expected product of 203 bp); AgRP, sense 5′GCA GAC CGA GCA GAA GAT GTT C3′and antisense 5′GTA GCA CGT CTT GAA GAA GC GG3′ (expected product of 186 bp); POMC, sense 5′CTC CTG CTT CAG ACC TCC ATA GAC3′ and antisense 5′AAG GGC TGT TCA TCT CCG TTG3′ (expected product of 164 bp) and CART, sense 5′TCT GGG AAG AAG AGG GAC TTT CGC3′and antisense 5′TCC ATT TGT GTT GCT TTG GGG TG3′ (expected product of 137 bp). All reactions were performed with an initial denaturation of 5 min at 95 °C, followed by 40 cycles of 30 s at 95 °C, annealing for 30 s at 55 °C, and extension for 1 min at 72 °C in an Mx3000 P qPCR System (Agilent Technologies). Ct values of GK mRNA obtained from three different experiments were normalized according to the 2−ΔΔCt method, using cyclophilin as a reference gene45.