Dietary induced obesity
All animals were provided by the Animal House of the Department for Experimental Medicine, Medical University of Silesia, Katowice, Poland and were treated in accordance to Directive 2010/63/EU for animal experiments using the protocols approved and monitored by the Local Ethics Committee for Animal Experimentation in Katowice. Animals were housed 4–5 per cage in a climate-controlled room (22 ± 2 °C, relative humidity: 55 ± 10%) with a 12 h:12 h light/dark cycle starting at 07:00 a.m.
28-day-old Long Evans male rats were used in two independent experiments. In Exp.1, animals were fed ad libitum with standard chow (control to obese cohort, ContO) or the same chow supplemented with Western diet foods (Obese). These included cheese, dry sausage made of pork (kabanos), crackers, salty potato chips, candy bars (bounty, mars, prince-polo) and a 10% saccharose solution to drink. Additionally, all animals had unlimited access to drinking water. After 10 weeks the animals were euthanized by decapitation to collect blood and heart samples.
In Exp. 2, animals from the AWL group (After Weight Loss) were treated in the same manner as in Exp. 1 to develop obesity, but this was extended for 2 more weeks (12 weeks of weight gain). After this time the rats were subjected only to standard rodent chow with an 80% calorie restriction (CR; the 100% of caloric needs was established empirically), which was reduced to 70% after two weeks until the end of the weight-loss-phase. During this period the control group from Exp. 2 (ContA) received the same isocaloric food to stop subtle weight gain in these control adult rats. The amount of food was established empirically for every cage and 20 g of standard chow/rat/day determined the criteria. After the period of weight reduction, the rats from AVL and ContA groups received an isocaloric amount of food for two weeks (stabilization phase) to equalize the calorie intake among control and experimental groups. Drinking water was provided ad libitum during all phases of Exp. 2.
Six hours prior to insulin tolerance and glucose loading tests the rats were fasted (tested animals from Exp.1 were fed by standard chow and water for 24 h before fasting), than insulin (1 U/kg in Exp.1; 0.75 U/kg in Exp.2; ActiRapid, Novo Nordisc) or glucose (2 g/kg) were injected in i.p. injections. Blood was collected from the tip of tail at the appropriate time intervals to measure glucose levels (CardioCheck Professional).
For the histological examination of the morphology of cardiomyocytes, 6 hearts per group collected during Exp. 1 and Exp. 2 were gently squeezed to remove excess blood, then weighed and immersed in a 10% formalin solution in PBS (pH = 7.2). For proteomic examination, fresh pieces of LV were collected from another animals (n = 5 per group in Exp. 1).
After decapitation ~ 2 mL of trunk blood was collected for serum and plasma samples. For the serum collection, the blood was allowed to clot by leaving it for 30 min on ice. The samples were then centrifuged (2,000 × g for 15 minutes, 4C), and the serum was pipetted and stored at − 80 °C. Serum cholesterol and triglyceride levels were measured in the sera using a Mindray BS-200 Chemistry Analyzer (Shenzhen Mindray Bio-Medical Electronics Co.).
For the plasma (used for proteomic profiling), 1 ml of blood was collected into 1.5 ml EDTA coated Eppendorf tubes containing 10 ul of 0.5 M EDTA and immediately centrifuged (1300 × g for 10 minutes, 4C). 99 ul of plasma was pipetted to fresh tubes containing 1 ul of a protease inhibitor cocktail (#P8340, Sigma Aldrich) [18].
Histological examination of the heart
The LV was dissected from formalin-fixed hearts. The tissues were dehydrated with graded concentrations of alcohol and embedded in paraffin. 5 µm paraffin slices from each tissue sample were stained with (1) Hematoxylin and Eosin (H&E) or (2) Masson’s trichrome stain.
To calculate the cross-sectional area of the cardiomyocyte, the sections stained with H&E were photographed (with 40x objective) using a Nikon light microscope (Nikon ECLIPSE E600) with an Olympus Camera (Olympus DP 26). The individual cell surface area was measured by a blinded observer using cellSens Entry Imaging Software (Olympus). One hundred cell surface areas were counted per each group, and the average value was used for analysis.
Masson’s stain was used to investigate LV morphology and perivascular/interstitial fibrotic changes. The sections were photographed (with 40x objective) 6 times each for the following: LV area without visible vessels (interstitial zone) and area with visible vessels (perivascular zone) [19]. The photos were subjected to deconvolution in the NIH Fiji program [20], than the areas of blue (connective tissue) or red (myocytes) channels were automatically counted. The data is expressed as the amount of connective tissue relative to the total area (connective tissue + myocytes) and expressed as a %.
Proteomic profiling
Protein extraction
The isolated brain tissues were lysed in a buffer with 1 M triethylammonium bicarbonate (TEAB) and 0.1% sodium dodecyl sulfate (SDS) and automatically homogenized using a Precellys 24 homogenizer (Bertin Technologies) in 0.5-mL tubes pre-filled with ceramic (zirconium oxide) beads (Bertin Technologies). Next, the material was subjected to a threefold cycle of freezing and thawing. Then, the tissue in the buffer was sonicated in a bath for three 1-minute cycles on ice and homogenized again using the Precellys 24 instrument. The protein concentration was measured using Pierce BCA protein assay kit (Thermo Fisher Scientific) in the isolated protein fraction according to the manufacturer’s instructions.
In-solution digestion
Ten-microgram aliquots of the proteins were diluted with 15 µl of 50 mM NH4HCO3 and reduced with 5.6 mM DTT for 5 min at 95 °C. The samples were then alkylated with 5 mM iodoacetamide for 20 min in the dark at RT. The proteins were digested with 0.2 µg of sequencing-grade trypsin (Promega) overnight at 37 °C.
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of the proteins
The analysis was performed with the use of the Dionex UltiMate 3000 RSLC nanoLC System connected to the Q Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific). The peptides derived from the in-solution digestion were separated on a reverse phase Acclaim PepMap RSLC nanoViper C18 column (75 µm × 25 cm, 2 µm granulation) using an acetonitrile gradient (from 4 to 60%, in 0.1% formic acid) at 30 °C and a flow rate of 300 nL/min (for 230 min). The spectrometer was operated in data-dependent MS/MS mode with survey scans acquired at a resolution of 70,000 at m/z 200 in MS mode, and 17,500 at m/z 200 in MS2 mode. The spectra were recorded in the scanning range of 300–2000 m/z in the positive ion mode. Higher energy collisional dissociation (HCD) ion fragmentation was performed with normalized collision energies set to 27.
Data analysis of the proteins
Protein identification was performed using the Swiss-Prot rat database with a precision tolerance set to 10 ppm for peptide masses and 0.08 Da for fragment ion masses. All raw data obtained for each dataset was imported into MaxQuant 1.5.3.30 version for protein identification and quantification. Protein was considered as positively identified if at least two peptides per protein were found by the Andromeda search engine, and a peptide score reached the significance threshold FDR = 0.01.
The obtained data was exported to Perseus ver. 1.5.3.2 software (part of the MaxQuant package). The numeric data was transformed to the logarithmic scale and each sample was annotated with its group affiliation. Next, the data was filtered based on valid values – proteins, which had valid values in 70% of samples in at least one group were kept in the table. A one-way analysis of variance (ANOVA) analysis was performed on the analyzed sample data with permutation-based FDR 0.05 used for truncation and the resulting list of differentiating proteins was normalized using a Z-score algorithm for the hierarchical clustering of data.
Gene ontology term analysis
Gene ontology (GO)-term enrichment analysis was performed with the DAVID functional annotation tool [21, 22]. The complete list of Rattus norvegicus proteins detected in mass spectrometry-based proteomics was used for the background and the GO term subcategories ‘GOTERM_BP_DIRECT’ ‘GOTERM_CC_DIRECT’ and ‘GOTERM_MF_DIRECT’ ‘INTACT’ were selected for analysis. An modified Bonferroni correction of p-value was applied to identify the statistically more represented function annotations
Immunoblotting
For immunoblotting, the samples were separated on 4–15% Stain-Free Gel (Bio-Rad) and transferred onto PVDF membranes. The membranes were blocked for 1 h at room temperature in Casein Blocking Buffer (Sigma-Aldrich) or 5% BCA, and incubated overnight at 4 °C (TBST + adequate blocking buffer) with primary antibodies produced in rabbits: anti-phospho AMPKα (Thr172) (Cell Signaling, #2535), anti-phospho mTOR (Thr2446 and Ser2448) (#15–105, MERK Millipore), anti-GLUT-4 (#PA1-1065, Invitrogen), anti-GLUT-1 (#ab652, Abcam), and anti-ACSL-1 (#PA5-17136, Invitrogen). After washing, the membranes were incubated with secondary donkey anti-rabbit IgG antibody (Abcam, ab205722). The immunoblots were visualized by means of Clarity Western ECL Blotting Substrates (Bio-Rad) and detected with the ChemiDoc™ Touch Imaging System (Bio-Rad). The targeted proteins were quantified with ImageLab Software 6.0.1 (Bio-Rad). The results were normalized to the total protein content in the gel (Stain-free technology, Bio-Rad).
Statistical analysis
Statistical analysis was performed using GraphPad Prism 8.01 software (GraphPad Software Inc.). Depending on the data distribution (as evaluated by the Shapiro-Wilk normality test), either the Student’s T-test or U-Mann-Whitney test were used for the estimation of significant differences between the two subject groups. In the case of repeating measurements, two-way ANOVA with Sidak’s multiple comparison test was applied. In all tests, significance was considered when p < 0.05.