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Glial cell derived neurotrophic factor prevents western diet and palmitate-induced hepatocyte oxidative damage and death through SIRT3


Animals

This study followed ARRIVE guidelines on the use of experimental animals. Animal studies were conducted in 5–6 weeks old female CF-1 (control) mice and GDNF transgenic (GDNF Tg) littermates. GDNF transgenic mice are on a CF-1 background and overexpress GDNF in cells expressing glial fibrillary acidic protein (GFAP) which is expressed in several tissues including the liver16,18. The mice were maintained on a 12 h light–dark cycle in a temperature-controlled barrier facility with free access to food and water. Four control (CF-1) mice and 4 GDNF Tg mice were assigned to a regular rodent diet (RD) (2018SX; Teklad Global 18% Protein Extruded Rodent Diet, Harlan Laboratories, Madison, WI; 6.2% fat by weight/18% kcal from fat) with regular drinking water and maintained on the diet for 25 weeks. Another 4 CF-1 mice and 4 GDNF Tg mice were assigned to a Western Diet (WD) (TD.120528, Harlan; 21.2% fat by weight/42% kcal from fat, with increased Sucrose, and 1.25% cholesterol) along with drinking water supplemented with a high sugar solution (FG) [23.1 g/L d-fructose (Sigma-Aldrich, cat. #1286504 USP) and 18.9 g/L d-glucose (Sigma-Aldrich, cat. #1181302 USP)]32. These mice were also maintained on the diet for 25 weeks. The complete composition of the diets is shown in Table 1. All animal studies were approved by the Atlanta Veteran Affairs Medical Center Institutional Animal Care and Use Committee and conducted according to the recommended guidelines.

Table 1 Rodent diet composition.

Cell culture

RALA255-10G rat hepatocytes were cultured in Gibco Dulbecco’s modified Eagle’s medium (DMEM) high glucose, HEPES, without pyruvate (#12430054, Life Technologies Corp, Grand Island, NY, USA) as previously described33. Human hepatocytes (H1000.H15B+, Lot No. HC3-37, Sekisui XenoTech, Kansas City, KS, USA) were cultured according to the vendors instructions. Cell culture media were replaced with fresh media every 48 h.

The rat and human hepatocytes were each assigned to 4 treatment groups: Vehicle, Palmitate, GDNF, and PA plus GDNF. Stock (6 mM) palmitate (Sigma-Aldrich, St. Louis, MO, USA) conjugated to fatty acid-free bovine serum albumin (BSA) (Sigma-Aldrich) was prepared as previously described34 and used at final concentration of 0.1–0.3 mM. Recombinant human and mouse GDNF (Shenandoah Biotechnology, Warwick, PA, USA) were used at a final concentration of 100 ng/mL.

Assessment of oxidative stress

Intracellular reactive oxygen species (ROS) levels were assessed in RALA255-10G rat hepatocytes cultured for 8 h in medium supplemented with or without palmitate (PA) and GDNF. The hepatocytes were stained with the general reactive oxygen species probe CM-H2DCFDA (Molecular Probes, Eugene, OR, USA) according to the manufacturer’s instructions and fluorescence measured on a SpectraMax iD3 Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA, USA). Lipid peroxidation was assessed in mice liver tissues using a Lipid Peroxidation (MDA) Assay Kit (#MAK085, Sigma-Aldrich).

Assessment of antioxidant and SIRT3 activity levels

Total cellular superoxide dismutase activity was assessed in RALA255-10G rat hepatocytes cultured for 24 h in medium supplemented with or without GDNF and palmitate using the Superoxide Dismutase (SOD) Colorimetric Activity Kit (# EIASODC, Life Technologies Corp. Frederick, MD, USA). SIRT3 deacetylase activity was also assessed in RALA255-10G rat hepatocytes cultured for 24 h in medium supplemented with or without GDNF and palmitate using a fluorometric SIRT3 Activity Assay Kit (# ab156067, Abcam, Waltham, MA, USA).

Assessment of mitophagic flux

Mitophagic flow was assessed by flow cytometry using a modification of a previously published protocol35. RALA255-10G rat hepatocytes were cultured for 6 h with or without palmitate (0.2 mM), GDNF (100 ng/mL), and with or without the lysosomal inhibitor chloroquine (CQ) (30 µM) (Cell Signaling Technology, Danvers, MA, USA). MitoTracker Red CMX-ROS (#M7512, Molecular Probes, Eugene, OR, USA) was then added to the cells at a final concentration of 25 nM in culture medium and the cells cultured for a further 15 min. The cells were washed once in PBS, stained for 30 min with LIVE/DEAD Fixable Near-IR Stain (#L34976, Life Technologies Corp., Carlsbad, CA, USA) and fixed with 3.7% formaldehyde in complete culture medium at 37 °C for 15 min. After washing 3 times with PBS with 1% bovine serum albumin, the cells were analyzed by flow cytometry at the Emory Pediatric and Winship Flow Cytometry Core (Emory University, Atlanta, GA, USA). Mitophagic flow was calculated from the difference in the number of MitoTracker Red CMX-ROS-positive hepatocytes between cells cultured in the presence of the inhibitor (CQ) and those cultured without CQ for each treatment.

Western blotting

Western blotting was performed as previously described17 using rabbit primary antibodies to SIRT3 (#5490, Cell Signaling Technology, Danvers, MA, USA), OPA1 (#80471, Cell Signaling Technology), cleaved caspase-3 (Asp175) (Cell Signaling Technology) and PINK1 (Sigma-Aldrich) diluted 1:1000. Mouse monoclonal primary antibodies to α-tubulin (DM1A) (Cell Signaling Technology) and β-actin (A5441, clone AC-15) (Sigma-Aldrich) were diluted, respectively, 1:1000 and 1:5000 before use. Horseradish peroxidase conjugated anti-mouse and anti-rabbit IgG (Cell Signaling Technology) secondary antibodies were used at 1:2,000 dilution. A semi quantitative measurement of band intensity was performed using the Carestream Molecular Imaging Software (Carestream Molecular Imaging, New Haven, CT, USA) and Fiji36.

Gene expression analysis

Total RNA was isolated using the RNeasy Mini kits (Qiagen GmbH, Hilden, Germany) and first-strand cDNA synthesized using SuperScript VILO (Invitrogen Life Technologies, Grand Island, NY, USA). Real-time PCR reactions were set up using Fast SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA) human SIRT3 upstream (5′-ATCGATGGGCTTGAGAGAGTGTC-3′) and downstream (5′-AACCCTGTCTGCCATCACGT-3′) primers and human GAPDH upstream (5′-AGCCTCAAGATCATCAGCAATGCC-3′) and downstream (5′-TGTGGTCATGAGTCCTTCCACGA-3′) primers. The primers were designed such that at least one primer in the pair spanned an intron to prevent it from priming on to genomic DNA. The inability of these primers to amplify genomic DNA was confirmed by PCR. Thermal cycling was performed on a StepOnePlus Real-Time PCR System (Applied Biosystems).

siRNA transfection

Rat hepatocytes were transfected with SMARTpool, ON-TARGETplus Rat Sirt3 siRNA (Catalog # L-084761-03-0005; Dharmacon, Cambridge, United Kingdom) or ON-TARGETplus Non-targeting Control Pool (Catalog # D-001810-10-05) using Lipofectamine RNAiMax (Invitrogen Life Technologies) according to recommended procedure.

Statistical analysis

Statistical analyses were conducted using the GraphPad Prism software version 3.00 for Windows (GraphPad Software, San Diego, CA). Data were tested for normality and subjected to unpaired t test or one-way ANOVA with Tukey posttest.



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