Animal models
Male C57BL/6 mice were purchased from Koatech and housed (one per cage) in individually ventilated cages under a 12-h light/dark cycle (lights on from 7:00 to 19:00) in a temperature- and humidity-controlled room with ad libitum access to water and normal-chow diet (NCD) (LabDiet, Inc., 38057) or HFD (60% kcal from fat; Research Diets, Inc., D12492). Food intake and body weight were observed daily just before the onset of the dark cycle as previously described [29]. The mice were divided into the following groups: control shRNA injected C57BL/6 mice group (shCon); shFgf11-expressing shRNA injected C57BL/6 mice group (shFgf11).
Generation of lentiviruses
Lenti-X 293 T cells (Clontech, 632180) were seeded on 100 mm dishes and transfected with psPAX2 packaging plasmid (6 μg), pMD2.G envelope plasmid (2 μg), and GPIZ constructs (8 μg, green fluorescent protein (GFP), carrying either control (Dharmacon, RHS4346) or Fgf11-targeting shRNA using TurboFect (Thermo Scientific, R0531) following the manufacturer’s instructions. To prevent off-target effects, 2 different shRNA plasmids targeting both A and B isoforms of Fgf11 (Dharmacon, VGM5520-200406248 and VGM5520-200407071) were selected after testing 6 different shRNA plasmids for mouse Fgf11. Culture medium containing lentiviruses was harvested and filtered through 0.45 μm syringe filters (Millipore, SLHV033RS) as previously described [29]. To obtain concentrated viruses, 4 successive rounds of ultracentrifugation were carried out in the same ultra-clear centrifuge tubes (Beckman, 344058) at 43,000 × g for 120 min at 4 °C [30]. After final centrifugation, pellets were gently resuspended in saline. The titers of lentiviral stocks were determined by flow cytometry [31]; that of control virus was 6.76 × 1010 and that of shFgf11-expressing lentivirus was 6.62 × 1010 IU/ml. The concentrated viruses were aliquoted and stored at − 80 °C.
Stereotaxic surgery
Seven-week-old C57BL/6 mice were acclimated for a week and were anesthetized with 10 ml/kg of body weight of a mixture of Zoletil, Rumpun, and saline 25 min before surgery. Lentiviruses were injected at a speed of 0.5 µl/min (1.32 × 108 IU/2 μl on each side) with a microliter syringe (Hamilton, 7768) using the following coordinates: 1.4 mm posterior to bregma; 6.2 mm ventral; 0.35 mm bilateral targeting the ARC [29].
Insulin tolerance test (ITT) and glucose tolerance test (GTT)
ITT was conducted 4 weeks after virus injection and GTT 5 weeks after virus injection. Mice were habituated to daily intraperitoneal injections of isotonic saline 3 days before each tolerance test. All procedures were started at 10:00 and performed with reference to general procedures [32, 33]. For ITT, 6 h-fasted mice were injected with 1.0 U insulin/kg (Sigma, Cat#I9278) and blood was collected from the tail vein at the designated times and used to measure glucose with a glucose monitor (Roche, Accu-Chek Active meter). GTT was performed using 16 h-fasted mice by an i.p. injection of 1.5 g/kg glucose (Sigma, Cat#G8270) and blood glucose was assessed as in ITT.
Determination of brown adipose tissue (BAT) temperature
To evaluate BAT thermogenesis, an infrared camera (FLIR E60, FLIR Systems, Inc.) was used with an intra-red resolution of 320 × 240 pixels. To rule out stress-induced thermogenesis, mice were neglected for an hour while being able to move freely. BAT temperature of each mouse was measured at least 3 times for each round, and the average temperatures from each of 5 rounds were used for analysis.
Determination of heat generation, O2 consumption (VO2), CO2 production (VCO2), respiratory exchange ratio (RER), and total locomotor activity
Two weeks after ARC Fgf11 knockdown with HFD feeding, indirect calorimetry was performed using metabolic chambers of Comprehensive Lab Animal Monitoring Systems (CLAMS; Columbus Instruments). Mice were housed individually with free access to water and HFD in metabolic chambers Mice were acclimated for 24 h before metabolic assessment. After acclimation, heat generation, VO2, VCO2, RER, and locomotor activity were measured using an Oxymax system (Columbus Instruments). VO2, VCO2, and heat production were assessed every 12 min during 24 h and were normalized to body weight; RER was calculated as VCO2/VO2. Locomotor activity was determined by measuring interruptions in the infrared beams (total X- and Z-beam breaks).
In situ hybridization
In situ hybridization for the simultaneous detection of Fgf11 and Npy in the ARC was performed using an RNAscope fluorescent multiplex kit (Advanced Cell Diagnostics; ACD). Brains were dissected from three mice, and were rapidly embedded in FSC 22 Frozen Section Media (Lecia, 3801480) and frozen on dry ice. Fresh frozen coronal Sections (20 μm) were cut on a cryostat (Lecia, CM3050S) and dual-labeled for the mRNA of Fgf11 (ACD, 701) and Npy (ACD, 313321-C2) following the manufacturer’s protocol. The Fgf11 antisense probe targeted the region 888–1891 of the mouse Fgf11 transcript variant 1 (NM_010198.3), but was cross-reactive with all the other transcript variants (NM_001291104.2, NM_001362623.1, NM_001362624.1). The Fgf11 sense probe (ACD, 843141) was used as a negative control, and the negative control probe (ACD, 320751) recognizing dihydrodipicolinate reductase, DapB (a bacterial transcript), was also used in parallel with the target probes. Fluorescent in situ hybridization images were taken using an LSM 780 or LSM 800 confocal laser-scanning microscope (Carl Zeiss) with maximal signal separation.
Immunohistochemistry
The processing, embedding, cryosectioning, and immunofluorescence staining of brain tissue were performed as previously described [34]. The final dilutions of primary antibodies—sheep anti-NPY (1:1000; Abcam, ab6173) and mouse anti-TH (1:1000; Immunostar, 22941)—were 1:1000. The following secondary antibodies were used (both at 1:500): Cy3-conjugated donkey anti-sheep IgG (1:500; Jackson ImmunoResearch, 713–165-147) and AlexaFluor 488–conjugated anti-mouse IgG (1:500; Jackson ImmunoResearch, 715-545-150). Sections were incubated for 5 min at room temperature with 1 μg/mL Hoechst 33342 (Invitrogen, H3570) in phosphate-buffered saline for nuclear staining, mounted on glass slides, and coverslipped with Vectashield Mounting Medium (Vector Laboratories, H-1000). From each mouse (at least 3 mice in total), 3–5 ARC or PVN sections were analyzed using LSM 780 or LSM 800 with maximal signal separation.
Measurement of immunofluorescence intensity
Standardized settings for image acquisition and processing intensity were performed for relative quantification of NPY and TH fluorescence. To obtain values for NPY immunofluorescence intensity in the ARC and PVN, morphological boundaries of each area were drawn on images. For TH immunofluorescence intensity in the TH neuron, the cell type-specific outlines were plotted with the corresponding gray-scaled TH immunofluorescence images. Single measurements of fluorescence intensity were performed using Image J software (National Institutes of Health, US). NPY fluorescence intensity for each mouse hypothalamic region were averaged from six independent measurements. TH fluorescence intensity for each TH neuron were averaged from four independent measurements. The fluorescence intensity of NPY and TH was plotted using arbitrary units ranging from 0 to 3. NPY-positive axon terminals adjacent within 1.5 µm from TH positive neurons were counted for counting the number of NPY-positive boutons.
Quantitative RT-PCR analysis of mRNA expression
Total RNA was isolated from cells and tissues using Trizol reagent (Invitrogen, 15596018). The RNA pellet was dissolved in nuclease-free water (Promega, P1193) and total RNA concentration was determined using a NanoDrop spectrophotometer (DeNovix, DS-11). Total RNA, reaction buffer, and GoScript Reverse Transcriptase (Promega, A5004) were mixed in a total volume of 20 μl and reverse transcription was carried out in a thermal cycler (Bio-Rad, C1000) at 25 °C for 5 min, 42 °C for 60 min, and 70 °C for 15 min. Real-time PCR was performed with a SYBR Green PCR kit (TaKaRa Biotechnology, RR820A) in a qPCR machine (Bio-Rad, CFX96) for 40 cycles (95 °C for 10 s, 60 °C for 30 s). The following primers were synthesized by Integrated DNA Technologies: Cart Forward, 5′-CGAGAAGAAGTACGGCCAAGTCC-3′; Cart Reverse, 5′-GGAATATGGGAACCGAAGGTGG-3′; Dio2 Forward, 5′-TGCCACCTTCTTGACTTT-3′; Dio2 Reverse, 5′-GTTTCCGGTGCTTCTTAACC-3′; Fgf11 Forward, 5′-TCGTCACCAAACTGTTCTGC-3′; Fgf11 Reverse, 5′-GCCATGTAGTGACCCAGCTT-3′; Gapdh Forward, 5′-ATCACTGCCACCCAGAAGAC-3′; Gapdh Reverse, 5′-ACACATTGGGGGTAGGAACA-3′; Npy Forward, 5′-CAGAAAACGCCCCCAGAA-3′; Npy Reverse, 5′-AAAAGTCGGGAGAACAAGTTTCATT -3′; Pgc1α Forward, 5′-AGCCGTGACCACTGACAACGAG-3′; Pgc1α Reverse, 5′-GCTGCATGGTTCTGAGTGCTAAG-3′; Pomc Forward, 5′-GAACAGCCCCTGACTGAAAA-3′; Pomc Reverse, 5′-ACGTGGGGGTACACCTTCAC-3′; Prdm16 Forward, 5′-CCGCTGTGATGAGTGTGATG-3′; Prdm16 Reverse, 5′-GGACGATCATGTGTTGCTCC-3′. Relative mRNA expression of each target gene was analyzed by the delta-delta Ct method and normalized to that of Gapdh.
Antibodies and chemical reagents
Target proteins were immunoblotted with the following antibodies: phospho-AKT (Ser473; Cell Signaling Technology [CST], 4060), AKT (CST, 9272), β-catenin active (CST, 8814), β-catenin (CST, 9582), phospho-CAMKII (Thr286; CST, 12,716), CAMKII (CST, 3362), phospho-CREB (Ser133; CST, 9198), phospho-CREB (Ser129; MyBioSource, MBS9406211), CREB (CST, 9197), phospho-ERK (Thr202/Thr204, 4370), ERK (CST, 9107), phospho-FOXO1 (Ser256; CST, 9461), FOXO (CST, 2880), GAPDH (CST, 2118), phospho-GSKα and β (Tyr279 and Tyr216; BD Biosciences, 612313), phospho-GSKα and β (Ser21 and Ser9; CST, 8566), GSKα and β (CST, 5676), HA-tag (CST, 3724), phospho-STAT3 (Tyr705; CST, 9145), and STAT3 (CST, 12640). When indicated, cells were treated with 2-deoxy-D-glucose (2DG; Sigma, D6134) to induce glucoprivation; 2DG was dissolved in DPBS (Corning, 21-031-CVR) or saline. 6-Bromoindirubin-3'-oxime (BIO; Sigma, B1686) was dissolved in dimethyl sulfoxide (Sigma, M81802).
Western blotting
Cells were lysed in 50 mM Tris–HCl, pH 7.4, 250 mM sucrose (Bioshop, SUC507), 5 mM sodium pyrophosphate, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100 (Sigma, T8787), 0.1 mM benzamidine (Sigma, B6506), 1 mM DTT, 0.5 mM PMSF (Sigma, P7626), 50 mM NaF, protease inhibitor cocktail (Calbiochem, 535,140), and phosphatase inhibitor cocktail (Sigma, P5726). Lysates were resolved in SDS–polyacrylamide gels and blotted onto PVDF membranes (Millipore, IPVH00010) for 35 min at 20 V in transfer buffer (25 mM Tris base, pH 7.4, 192 mM glycine, 10% methanol). The membranes were blocked with 5% skim milk for 1 h and incubated with appropriate primary antibodies for 1 h at room temperature or at 4 °C overnight. After 3 washes with TBST buffer (20 mM Tris [Bioshop, TRS001], 125 mM NaCl [Bioshop, SOD001], 0.1% Tween 20 [Sigma, P1379]), each membrane was incubated with appropriate HRP-linked secondary antibody (anti-mouse: CST, 7076S; anti-rabbit: Thermo Scientific, NCI1460KR) and the bands were visualized by using ECL solutions (Thermo Scientific, NCI4080KR; Advansta, K-12045-D50) according to the manufacturer’s instructions. Band intensities were measured and quantified using ImageJ software.
Cell lines
The embryonic mouse hypothalamic mHypoE-N41 (N41; Cellutions Biosystems Inc., CLU121) and mHypoE-N43/5 (N43/5; Cellutions Biosystems Inc., CLU127) cell lines were maintained in DMEM (Sigma, D5796) with 10% fetal bovine serum (Hyclone Laboratories Inc., SH30919.03) and 1% penicillin/streptomycin (Hyclone Laboratories Inc., SV30010) at 37 °C.
2DG and 0-mM glucose treatment
For glucoprivation (2DG) or glucose deprivation (glucose-free), 2DG was added to 25-mM glucose DMEM (Welgene, LM001-07) or 0-mM glucose DMEM (Welgene, LM001-56), was used, respectively. Lenti-X 293T (Clontech, 632180) cells were maintained in DMEM (Hyclone Laboratories Inc., SH30243) under the same conditions as hypothalamic cell lines.
siRNA transfection
N41 and N43/5 cells were seeded in 6-well plates and transfected with ON-TARGETplus mouse scrambled or Fgf11 siRNA comprised of 4 different siRNAs. Scrambled siRNAs (100 nM; Dharmacon, D-001810–10-10–05) or Fgf11 siRNAs (100 nM; Dharmacon, L-045551–01-0010) were transfected using Lipofectamine 3000 for 48 h following the manufacturer’s instructions.
Statistical analysis
All data were reported as means ± standard error of the mean (SEM). Statistical significance was determined by two-tailed t-test or two-way analysis of variance (ANOVA) followed by a Tukey multiple comparison test using GraphPad Prism 8; p values < 0.05 were considered statistically significant.