Animals
All animal procedures where performed in accordance with institutional regulations at the University of Colorado School of Medicine. Male NEXLPL-/-, NEXLPL+/- and WT mice were generated as previously described and housed in standard conditions, fed standard laboratory chow diet until terminal experiments at 3 mo. (WT n= 4, NEXLPL-/- n=4), 6 mo. (WT n= 8, NEXLPL-/- n=6, NEXLPL+/- n =6), 12 mo (WT n= 3, NEXLPL-/- n=5, NEXLPL+/- n =5), 18 mo. (WT n= 6, NEXLPL-/- n=5, NEXLPL+/- n =6) (11). Mice were fasted for four hours prior to tissue harvest. Body composition was measured on anesthetized mice by dual-energy x-ray absorptiometry using a mouse densitometer (PIXImus2, Lunar Corp., Madison, WI). The electrophysiological studies were conducted at Tulane University. The procedures were approved by the Institutional Animal Care and Use Committee.
Glucose and insulin tolerance tests
Glucose and insulin tolerance tests were performed by bolus intraperitoneal injection of glucose (1 g/kg) or insulin (0.75 units/kg), respectively. Blood glucose was measured from the tail using a glucometer (OneTouch Ultra, Lifescan) at baseline (0) and 10, 20, 30, 45, 60, and 90 min after injection.
Hyperinsulinemic-euglycemic clamps
Before the clamp experiment, mice were fasted overnight. On the day of the clamp experiment, mice were anesthetized and an indwelling catheter was inserted in the right internal jugular vein (14). A three-way connector was attached to the catheter to intravenously deliver solutions. A 2-hour hyperinsulinemic-euglycemic clamp was conducted in all four groups of mice with a primed (150 mU/kg body wt) and continuous infusion of insulin (Humulin; Eli Lilly, Indianapolis, IN) at a rate of 2.5, 5 or 10 mU/kg/min to raise plasma insulin within a physiological range. 20% glucose was infused at variable rates to maintain glucose at basal concentrations. Blood samples (10 µl) were collected at 10 min intervals for measurement of plasma glucose concentration only. Basal and insulin stimulated whole-body glucose turnover were estimated with a continuous infusion of [3-3H]glucose (PerkinElmer, Boston, MA) for 2 h before the clamps (0.05 µCi/min) and throughout the clamps (0.1 µCi/min), respectively. To estimate insulin-stimulated glucose uptake in individual organs, 2-deoxy-D-[1- 14C] glucose (2-[14C]-DG) was administered as a bolus (10 µCi) when euglycemic clamp has approached. 10 min after 2-[14C]-DG being injected, tissues were taken for biochemical analysis.
Analysis of liver lipids
Neutral lipid was analyzed as described previously (15-17). Frozen liver tissue was homogenized in (v/v) Folch reagent (2:1 CHCl3/MeOH) containing 300 mg of tritridecanoin reference standard (Nu-Check Prep Inc., Elysian, MN) by bead homogenization for two cycles of 2 min at 30 Hz. Homogenates were diluted further with Folch reagent to 4 ml, treated with 800 ml of 0.9% sodium chloride solution, vortexed, and centrifuged at 4000 rpm for 5 min. The organic phase was removed and dried under N2 gas. Total lipids were resuspended in 330 ml of 100% chloroform and applied to HyperSep SI SPE columns (Thermo Scientific, Waltham, MA) pre-equilibrated with 15 column volumes chloroform. Neutral lipids were eluted with a total of 3 ml of chloroform, dried under N2, and resuspended in 1 ml of methanol containing 2.5% H2 SO4 . Fatty acid methyl ester (FAME) production was initiated by heating at 80 °C for 1.5 h. 1 ml of HPLC-grade water was added to quench the reactions, and FAMEs/cholesterol was extracted with 200 ml of hexane. A Trace 1310 GC with a TG-5MS column (Thermo Scientific, Waltham, MA) was used to separate lipids chromatographically, and lipids were analyzed with an ISQ single quadrupole mass spectrometer. Xcalibur software (Thermo Scientific) was used to calculate peak areas. Areas were normalized to the tritridecanoin reference standard and then to tissue weight. Histological analysis and scoring of hepatic lipids and steatosis were performed as previously described (18).
Lipoprotein Profile
Plasma samples (200 µL) were chromatographed via fast protein liquid chromatography (FPLC) using two Superose 6 columns in series as previously reported (19). Cholesterol was measured in each fraction using a commercially available kit (Cayman Chemical Company, Ann Arbor, MI, USA) following procedures outlined in the package insert.
Quantitative real-time PCR
Total RNA was reverse transcribed with the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA). Quantitative PCR was performed using primer sets for genes of interest and reference genes (designed using NCBI’s Primer3/BLAST) and iTaq Universal SYBR Green Supermix (Bio-Rad) following manufacturer's protocols. Reactions were run in duplicate on an iQ5 Real Time PCR detection system (Bio-Rad) along with a no-template control per gene. Validation experiments were performed to demonstrate that efficiencies of target and reference genes were approximately equal. Data were normalized to two reference genes (GAPDH and ACTB) using the comparative Ct method.
Identification of liver-related neurons with PRV-152
Retrogradely transported pseudorabies viral vector (PRV-152, provided by the Center for Neuroanatomy with Neurotropic Viruses) expressing enhanced green fluorescent protein (EGFP) was used to identify liver-related neurons (20-22). Under anesthesia, the liver was exposed with a small transverse incision and ~4 ml of PRV-152 was injected into the median lobe of the liver (2 injections of 2 sites). A drop of adhesive “liquid bandage” was used to seal each injection to prevent the leakage of the virus. The animals were maintained in a biosafety level 2 facility up to 110 h post-injection.
Brain slice preparation
Acute brain slices were prepared from WT and NEXLPL-/- mice. After anesthesia with isoflurane, the brain was removed and immersed in ice-cold oxygenated artificial cerebrospinal fluid (aCSF) containing the following (in mM): 124 NaCl, 26 NaHCO3, 1.4 NaH2PO4, 11 glucose, 3 KCl, 1.3 MgCl2, 1.5 CaCl2, pH 7.3-7.4. Transverse hypothalamic slices containing the PVN (300mm) were made using a vibrating microtome. The slices were stored in a holding chamber at 34-36°C, and then transferred to a recording chamber mounted on a fixed stage under an upright microscope (Nikon FN1).
Whole-cell patch-clamp recordings
Whole-cell patch-clamp recordings were performed at 34-36°C from liver-related neurons in the PVN identified under 40x water-immersion objective (N.A=0.8). Epifluorescence was used to identify EGFP-containing neurons and infrared illumination and differential interference contrast optics (IR-DIC) to target specific cells. For whole-cell patch-clamp recordings, electrodes (3-7 MW) were filled with a solution containing the following (in mM): 130 Cs+ gluconate, 10 HEPES, 5 EGTA, 1 NaCl, 1 MgCl2, 1 CaCl2, 3 CsOH , 2-3 Mg-ATP, 0.2% biocytin, pH 7.3-7.4. Electrophysiological signals were recorded using an Axoclamp 700B amplifier (Molecular Devices) and acquired by pClamp (Molecular Devices). Inhibitory post-synaptic currents (IPSCs) were recorded at -10 mV and excitatory post-synaptic currents (EPSCs) at -60 mV. Data were analyzed offline using pClamp or MiniAnalysis (Synaptosoft).
Stereotaxic AVV injection
Adeno-associated virus 8 coding for Cre recombinase and GFP under control of CaMKIIa promoter (AAV8-CaMKIIa-GFP-Cre, UNC viral core) was utilized to achieve recombination between flox sites of PVN neurons. Adeno-associated virus 8 coding for GFP under control of CaMKIIa promoter (AAV8-CaMKII-GFP, UNC viral core) was used as control vector (mice referred to as PVN nLPL+). 10-week old male LPL flox/flox mice were anesthetized with 2.0% isoflurane and surgical anesthesia plane was maintained with 1.0% isoflurane. Incision site was locally anesthetized with 100-200 μl of lidocaine (1 mg/mL). Mice were then mounted onto stereotaxic apparatus (Neurostar). 100 nL of virus was injected bilaterally into the PVN (anteroposterior −0.7 mm, lateral +/- 0.25 mm, and dorsoventral −4.8 mm) using 0.5 μl Hamilton syringe (part# 86250, Hamilton Company) at a rate of 20 nL/min (23). Syringe was kept in place for 5 minutes after each infusion and the needle was withdrawn over 3 minutes. 2 weeks following the initial injection, anesthetized mice were transcardially perfused with HBSS (with calcium and Magnesium) and whole brains were fresh-frozen in liquid nitrogen-cooled 2-methylabutane, since fixed brains are incompatible with Fluorescence Lifetime Imaging Microscopy.
Fluorescence Lifetime Imaging Microscopy (FLIM)
FLIM was performed to detect local metabolic changes in 5-7 different areas of PVN in a fresh brain sections using a Zeiss 780 laser-scanning confocal/multiphoton-excitation fluorescence microscope with a 34-Channel GaAsP QUASAR Detection Unit and non-descanned detectors for 2-photon fluorescence (Zeiss, Thornwood, NY) equipped with an ISS A320 FastFLIM box and a titanium:sapphire Chameleon Ultra II (Coherent, Santa Clara, CA). The 2-photon excitation was blocked by a 2-photon emission filter. For the acquisition of FLIM images, fluorescence for Nicotinamide Adenine Dinucleotide (NADH) and Flavin Adenine Dinucleotide (FAD) was detected simultaneously by two photon-counting PMT detectors (H7422p-40; Hamamatsu). Images of the different areas of PVN in the brains were obtained with Vista Vision software by ISS in 256x256 format with a pixel dwell time of 6.3 µs/pixel and averaging over 30 frames. Calibration of the system was performed by measuring the known lifetime of the fluorophore fluorescein with a single exponential decay of 4.0 ns (24). The phasor transformation and data analysis were carried out using Global SimFCS software (Laboratory for Fluorescence Dynamics (LFD), University of California, Irvine) as described previously (25). The number of pixels covered with lifetimes for free and bound reduced form of NADH and FAD were calculated in SimFCS (LFD) and the values were normalized to the total number of pixels detected as previously described (26, 27).
The glycolytic index was calculated for all experimental groups using the following equation: as defined previously (27, 28).
Cell Culture conditions and Reagents
mHypoE41 (N41) immortalized mouse hypothalamic neurons were purchased from CELLutions Biosystems (Winnipeg, MB). N41 cells were grown in DMEM containing 1000 mg/L glucose and 10% FBS at 37°C in the presence of 5% CO2. To produce cells for stable overexpression of LPL (N41 mLPL or Empty [control]), N41 cells were transduced with MSCV as previously described (10). To produce stable knock-down cells, N41 cells were transduced with shRNA (N41 553, or N41 202 [control]) containing lentivirus as previously described (10).
Metabolomics
Frozen cell pellets were extracted at 2e6 cells/mL in ice-cold lysis/extraction buffer (methanol:acetonitrile:water 5:3:2 v/v/v). Metabolites were separated using a 9-minute C18-based gradient method as described (29), using a Thermo Vanquish UHPLC coupled to a Thermo Q Exactive mass spectrometer. Amino acids (30), and other metabolites (31), were quantified as previously described.
Statistical analysis
For electrophysiological experiments, continuous recordings have been conducted and 2-minute periods were analyzed with MiniAnalysis (Synaptosoft) to measure peak amplitude and frequency of post-synaptic currents. Comparison between groups was made with an unpaired two-tailed Student’s t test. For all analysis, P<0.05 was considered significance. Numbers are reported as mean ± standard error of mean (SEM).
Two-way repeated measure ANOVA was performed for all age and time related analysis, using post-hoc multiple comparison with Bonferroni correction, using GraphPad 7 data analysis and graphing software, with P<0.05 being considered significant and P<0.1 a trend.