Animal models
Six-week-old BALB/c female mice (The Charles River Laboratories, Italy) were housed in sterile enclosures under specific pathogen-free conditions. The 30 mice were divided into two groups: with 15 animals, the “control” group were kept under standard conditions and exposed to fasting for 6 h before the study; the remaining animals were submitted to 48 h of starvation (“STS,” absence of food and free access to water) before imaging. In each group, nine mice were submitted to micro-PET imaging while six mice were sacrificed for the ex vivo studies and thus for measurement of FDG uptake (n = 3) or biochemical analyses (n = 3), respectively.
Experimental micro-PET scanning protocol
In vivo imaging was performed according to our validated procedure [
14]. Anesthesia was induced by intraperitoneal administration of ketamine (100 mg/kg) and xylazine (10 mg/kg). Capillary glucose level and body weight were measured, and mice were positioned on the bed of a dedicated micro-PET system (Albira, Bruker Inc., USA). A dose of 3–4 MBq of FDG was then injected through a tail vein, soon after the start of a list mode acquisition lasting 50 min.
Image processing
List data were divided according to the following framing rate: 10 × 15 s, 5 × 30 s, 2 × 150 s, 6 × 300 s, 1 × 600 s, and then reconstructed using a maximal likelihood expectation maximization method (MLEM). Two nuclear doctors unaware of mouse allocation drew a volume of interest (VOI) in the left ventricular chamber to plot the time-concentration curve in arterial blood throughout the whole acquisition (input function). Whole body FDG clearance (in ml × min
−1) was calculated using the conventional stochastic approach as the ratio between injected dose and integral of input function, fitting the last 20 min with a mono-exponential function [
15]. In vivo CMRGlu* was estimated according to Gjedde-Patlak [
16] graphical analysis by using the routine of dedicated software (PMOD, Zurich, Switzerland) with lumped constant value set at 1. On these parametric maps, two VOIs were drawn to estimate the average brain (CMRGlu*) and skeletal muscle (SM-MRGlu*) in nMol × min
−1 × g
−1. These same VOIs were thus transferred on the last 600-s frame to estimate FDG standardized uptake value (SUV). According to the same procedure, at regional analysis, cortical and cerebellar CMRGlu* and SUV were estimated to calculate the cortical/cerebellum ratio in the two studied subgroups of animals.
Ex vivo experiments
For “ex vivo” evaluation, each brain was harvested soon after sacrifice, stuck in the outer ring of a Petri dish with octyl-cyanoacrylate (Dermabond, Ethicon, USA), and covered with 2 mL solution collected from an input vial containing 3 mL of DMEM medium (12.5 mM glucose) with a known FDG concentration (1 MBq/mL). Time-activity curve (TAC) of tracer uptake was thus plotted using the Ligand Tracer White device (Ridgeview, Uppsala, Se) [
17,
18]. Briefly, this instrument consists of a beta-emission detector and a rotating platform harboring a standard Petri dish. The rotation axis is inclined at 30° from the vertical, so that the medium covers the dish nadir while the detector points at its zenith. All experiments consisted of 45 periodic rotations lasting 1 min and divided into four intervals: (
a) brain kept for 25 s in the system nadir and thus fully immersed in the incubation medium, (
b) 5-s 180° counter-clockwise rotation, (
c) brain kept for 25 s under the detector at the system zenith and, finally, and (
d) 5-s 180° counter-clockwise rotation for cycle restart. At each cycle, the detector measures background and target counting rates (in counts per second, CPS) in phases
a and
c, respectively. FDG brain TAC was thus obtained by subtracting the background counting rate from the corresponding target value [
19].
At the end of the experiment, an aliquot of 0.5 mL was sampled both from input vial and from Petri dish (output) to measure glucose concentration (mM) and total FDG activity (MBq). Brain TAC was thus normalized by multiplying brain counting rate at each time
t (BCR(t)) for the following factor:
$$ \mathrm{BFD}(t)=\mathrm{BCR}(t)\times \frac{1}{{\mathrm{BCR}}_{\left(45\ \min \right)}}\times \frac{\left({A}_{\mathrm{input}}-{A}_{\mathrm{output}}\right)\kern0.50em }{A_{\mathrm{input}}} $$
(1)
where BFD(
t) represents the fraction of the dose present in the brain at each time
t, BCR
(45 min) represents the brain counting rate in the last minute,
Ainput and
Aoutput represent FDG activity in MBq at experiment start and end, respectively.
The closed system nature of the Ligand tracer permitted us to consider the input function (IF) as:
$$ \mathrm{IF}(t)=1-\mathrm{BFD}(t) $$
(2)
BFD(
t) and IF(
t) were thus used according to Patlak graphical analysis, assuming the volume invariance of both incubation medium and the brain, during the experiment, respectively. The regression line was defined as:
$$ \frac{{\mathrm{BFD}}_t}{{\mathrm{IF}}_t}=a\ \frac{\int_0^t{\mathrm{IF}}_t dt}{{\mathrm{IF}}_t}+b $$
(3)
This curve was analyzed in order to verify the expected accumulation kinetics of FDG; the slope
a was identified by least squares’ definition of regression line and multiplied for input glucose level to estimate CMRGlu*, with the star denoting the FDG-based measurement of CMRGlu, according to the original definition of Sokoloff et al. [
20]. By contrast, ex vivo CMRGlu (in nMol × min
−1) was measured by the equation:
$$ \mathrm{CMRGlu}=\left({\mathrm{Glucose}}_{\mathrm{input}}-{\mathrm{Glucose}}_{\mathrm{output}}\right)\kern0.5em \left(\frac{\mathrm{nanoMol}}{\mathrm{mL}}\right)\times \kern0.5em \frac{2}{45}\kern0.5em \left(\frac{\mathrm{ml}}{\min}\right) $$
(4)
where glucose represents glucose concentration (nM), 2 is the volume of used DMEM, and 45 is the experiment duration.
Ex vivo imaging
Soon after the end of the ex vivo experiment, brains were washed and frozen in isopentane chilled with dry ice for sectioning with a cryomicrotome in slices 100 μM thick. At least three sections per brain were placed on a microscope slide and exposed to an imaging plate (Cyclone, PerkinElmer, USA) that provides an image resolution of 100 μm. Exposure time was optimized to 5 min. Thereafter, brain sections were stained with hematoxylin/eosin and photographed by inverted optical microscope. No measurement of radioactivity content was attempted, while autoradiography images were co-registered with the histologic staining using ImageJ software.
Brain homogenate analysis
For biochemical analyses, brains were homogenized in phosphate-buffer saline (PBS) solution with a Potter-Elvehjem homogenizer. Proteins concentration was performed by Bradford analysis [
21]. The samples were sonicated for 10 s in ice.
Western blot experiments were performed accordingly to the standard procedure using 50 μg proteins for each sample. Enzymatic assays were performed spectrophotometrically in a double-beam spectrophotometer (UNICAM UV2, Analytical S.n.c., Italy) using 100 μg of protein for each sample.
Activities of hexokinase (HK), phosphofructokinase (PFK), glucose-6-phosphate dehydrogenase (G6PD), G6Pase, and Complex I (NADH-ubiquinone oxidoreductase) were assayed according to the methods in our previously validated procedure [
14]. β-Hydroxy-butyrate-dehydrogenase (BHBDH) activity was evaluated following reduction of NAD
+ at 340 nm using a solution of 200 mM Tris-HClpH 8, 2 mM NAD
+, and 30 mM β-hydroxybutyrate. Gluthatione reductase activity was evaluated spectrophotometrically, at 405 nm, using Glutathione Reductase Assay Kit (Abcam: ab83461) following the manufacturer’s instructions. Real-time PCR evaluation was performed according to the standard procedures of our lab [
22].
Value Km and Vmax for HK
Hexokinases (HK) Michaelis-Menten kinetics was evaluated for in the presence of glucose and 2-deoxyglucose (2DG; Sigma-Aldrich, Saint Louis, MO, USA). The kinetic characterization of HK was determined at pH 7.4 and 25 °C, by coupling hexose phosphorylation to the reduction of NADP, recording the change in absorbance at 340 nm. The studied initial concentrations were 0.05, 0.1, 1, 5, and 200 mM for glucose and 0.3, 5, 50, 100, and 200 mM for 2DG. To avoid the substrate selectivity, G6PD was substituted with hexose-6P-dehydrogenase (H6PD), as an enzyme able to process both hexoses. Vmax (the maximum rate achieved by the system) and Km (the Michaelis-Menten constant indicating the substrate concentration at which the reaction rate is Vmax/2) were determined by Lineweaver-Burk double reciprocal plots.
Statistical analysis
Data are presented as mean ± standard deviation (SD). For comparison between different groups, the null hypothesis was tested by Student’s t test for paired or unpaired data, as appropriate. Significance was considered for p values p < 0.05. Statistical analyses were performed using SPSS software 15.0 (Chicago, IL, USA).