Fasting-induced G0/G1 switch gene 2 and FGF21 expression in the liver are under regulation of adipose tissue derived fatty acids

Graphical abstract


Animals and diets
CGI-58-floxed mice [1,2] and Fab4-Cre transgenic mice [3] have been previously described and used for the generation of mice with AT-selective disruption of CGI-58 (CGI-58ATko). Littermates homozygous for the floxed CGI-58 allele (flox/flox) were used as controls. Atgl-floxed mice [4] and AdipoQ-Cre transgenic mice [5] have been used for the generation of mice with AT-selective disruption of ATGL (ATGL-ATko). Mice were housed in a pathogen-free animal facility on a regular 12-h light/dark cycle and had free access to food and water. 14-to 16-week-old controls and CGI-58ATko mice were fed a standard chow diet (Ssniff) or subjected to a high fat diet containing 45% calories from fat (Ssniff) starting at 5 weeks of age for the duration of 10 weeks. Animals were subjected to 6-h-fasting or overnight-fasting (16-h-fasting) and then sacrificed.
For the administration of the PPARα agonist Wy14643 (Cayman Chemical), 12-h-fasted control and CGI-58ATko mice were i.p. injected either the Wy14643 compound (50 µg/g body weight in 50% DMSO) or equivalent amounts of vehicle (50% DMSO in 0.9% NaCl). After another 2-h-fasting period, mice were sacrificed and tissues were excised.
Animal care and study protocols were approved by the Austrian ethics committee and were in accordance with the Council of Europe Conventions.
For raising plasma FA levels, mice were fasted for 12 h and were then administered an intragastric olive oil gavage (200 µl/mouse). To release lipoprotein lipase (LPL) from the luminal site of capillaries and to hydrolyze TG-rich lipoproteins, heparin was administered i.p. (10 IU/mouse). 2 h after oil-gavage/heparin, mice were anaesthetized and blood was collected from the retro-orbital sinus for subsequent plasma analysis.
Thereafter, mice were euthanized by cervical dislocation and tissues were harvested and snap-frozen until further qRT-PCR analysis.

Gene expression analysis by quantitative real-time PCR
Total RNA was isolated with TRIzol reagent (Invitrogen) and treated with DNase I (Invitrogen). One µg total RNA was reverse transcribed using the MultiScribe High Capacity Reverse Transcription kit (Applied Biosystems) at 37°C for 2 hours.  Table S1.

Xbp1-PCR
1µl of liver cDNA was used as template and the PCR was performed applying 3 min 94°C, 30 cycle of 30 sec at 94°C, 30 sec at 58°C, 30 sec at 72°C and finally 3 min at 72°C. PCR products were run on a 3% agarose gel.

Plasma parameters and HOMA-IR
Blood samples were collected from shortly anesthetized non-fasted, 6-h-and 16-hfasted mice by retro-orbital puncture. Plasma TG, FA, glycerol, hormone and cytokine levels were determined using commercially available kits. The homeostasis model for insulin resistance (HOMA-IR) was calculated from 6-h-fasting glucose (mmol/L) x fasting plasma insulin (µU/ml) divided by 22.5.Plasma levels for insulin, FGF21, leptin, ketone bodies and levels of plasma growth hormone were analyzed using ELISA or Colorimetric Assay kits from Crystal Chem. (Rat Insulin ELISA kit), Cayman Chemical (ß-Hydroxybutyrate), Millipore/Merk (Rat/Mouse FGF-21 ELISA). Adiponectin levels were determined using the Mouse/Rat Adiponectin ELISA Kit (B-Bridge International, Inc.,CA, USA). Mouse TNF alpha and IL-6 were measured using the ELISAs "Ready-SET-Go!" from eBioscience.

Plasma ALT
ALT activity was determined in plasma of mice using the Infinity ALT(GPT) Liquid stable reagent according to the manufacturer's protocol. Briefly, 20 µl of plasma were incubated with 200 µl of ALT reagent for a total time of 10 min at 37°C in a Beckman DU640 spectrophotometer. The reaction is monitored by measuring the rate of the decrease in absorbance at 340 nm per minute.

Plasma non-saturated and saturated FA species
Analysis of plasma FA species from overnight fasted control and CGI-58ATko mice was performed as previously described [6].

Measurement of tissue TG hydrolytic activities
Tissue neutral TG hydrolase activity in the absence and presence of recombinant CGI-58 was performed as previously described [7,8]. Briefly, tissue of 16-h-fasted animals was homogenized in Solution A (0.25 M sucrose, 1 mM EDTA, 1 mM DTT, 1 µg/ml pepstatin, 2 µg/ml antipain, 20 µg/ml leupeptin, pH 7.0) on ice using an Ultra Turrax (IKA) and the infranatant after centrifugation at 20,000 x g and 4°C for 30 min was assayed for TG hydrolase activity. A micellar substrate of phospholipid-emulsified triolein (1.67 mM) containing [9,10-3 H]-labeled triolein (Perking Elmer) was used. Tissue lysates in a total volume of 100 µl solution A were incubated with 100 µl substrate in a shaking water bath at 37°C for 60 min in the absence or presence of the ATGL-specific inhibitor ATGListatin (20 µM) [9]. The reaction was terminated by the addition of 3.25 ml methanol/chloroform/n-heptane (10/9/7, v/v/v) and 1 ml 0.1 M potassium carbonate/0.1 M boric acid, pH 10.5. After centrifugation, radioactivity in the upper phase was determined by liquid scintillation counting.

Measurement of tissue TG levels
Tissue lipids were extracted according to the method of Folch et al [10]. Tissue lipid extracts were dried using a SpeedVac (Heto) and re-solubilized by brief sonication in 2 Remaining tissue pieces were lysed in lysis buffer (0.3 N NaOH/0.1 % SDS) and protein concentration was determined using the BCA Protein Assay Kit (Pierce, Thermo Scientific).

Pyruvate tolerance tests
After an overnight fast (16 h), mice were injected with sodium-pyruvate (2g/kg body weight) dissolved in saline and glucose blood glucose levels were determined after 15, 30, 60, 90 and 120 min.

Western Blot analyses
Tissues of 16-h-fasted mice were homogenized in Solution A (0.25 M sucrose, 1 mM EDTA, 1 mM DTT, 1 µg/ml pepstatin, 2 µg/ml antipain, 20 µg/ml leupeptin, pH 7.0) on ice using an Ultra Turrax (IKA). Tissue lysates were centrifuged at 20,000 x g and 4°C for 30 min and the infranatants were assayed for protein concentration using the Bio-Rad protein assay (Bio-Rad Laboratories) and BSA as standard.
Nuclear extracts were prepared from frozen liver samples either by the use of the NE-PER Nuclear and Cytoplasmic Extraction Reagents following the manufacturer's instructions (Pierce, Thermo Scientific) or by using a protocol modified from Lamond Lab Protocol. Briefly, tissue pieces were homogenized in Buffer A (10 mM HEPES pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 1 µg/ml pepstatin, 2 µg/ml antipain, 20 µg/ml leupeptin) using a Wheaton tissue grinder (Thermo Scientific). The homogenate was filtered through a cell strainer (70 µm, Corning) and centrifuged at 228 x g and 4°C for 10 min. The supernatant was withdrawn containing cytosolic proteins. Subsequently, the pellet was resuspended in Buffer A containing 1 % NP-40 using a tissue grinder and centrifuged at1,000 x g and 4°C for 10 min. After rinsing in Buffer A, the pellet was resuspended in sucrose solution S1 (0.25 M sucrose, 10 mM MgCl 2 ) and layered over sucrose solution S3 (0.88 M sucrose, 0.5 mM MgCl 2 ). After centrifugation at 2,800 x g and 4°C for 10 min, the nuclear pellets were sonicated twice for 10 seconds in RIPA buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1 % NP-40, 0.5 % deoxycholate, 1 µg/ml pepstatin, 2 µg/ml antipain, 20 µg/ml leupeptin) using a Misonix Ultrasonic Liquid Processor (QSonica). The supernatant obtained after centrifugation at 2,800 x g and 4°C for 10 min was collected as nuclear extract and assayed for protein concentration using the BCA Protein Assay Kit (Pierce, Thermo Scientific) and BSA as standard. For analysis of hepatic insulin sensitivity, 6-h fasted mice were injected with insulin (1U/kg body weight) and after 10 min livers were dissected for determination of phosphorylated (pSer473) and total Akt using antibodies from EMD Millipore. Levels were normalized to ß-Actin and pAkt/Akt ratios were calculated.
Bound immunoglobulins were detected using horseradish peroxidase-conjugated antirabbit, anti-mouse or anti-goat IgG antibody (Vector laboratories, GE Healthcare, Millipore) and visualized by enhanced chemiluminescence detection (ECL Plus Western blotting substrate, Pierce, Thermo Fisher Scientific). Quantification of signal intensities was performed using the ImageJ software.

Acute cold exposure
For acute cold exposure experiments, non-fasted and 10 hours fasted mice were singlehoused and exposed to 4°C. Before the start of the experiment and at indicated time points rectal body temperature was measured by the usage of a Temp10T Thermocouple thermometer (Thermo Scientific, Waltham, MA) and a Ret-3 rectal probe for mice (Physitemp, Clifton, NJ). Supplementary Fig. 1: (A)

Supplementary table 1
Plasma cytokine and adiponectin levels in fasted flox/flox and CGI-58ATko mice on HFD.