Glial cell-derived neurotrophic factor inhibits Western diet and palmitate-induced oxidative damage to hepatocytes and death through SIRT3.

the animals

This study followed ARRIVE’s guidelines for the use of experimental animals. Animal studies were conducted in 5- to 6-week-old CF-1 (control) female mice and GDNF transgenic littermates (GDNF Tg). GDNF transgenic mice are present on a CF-1 background and overexpress GDNF in cells expressing glial fibrillary acidic protein (GFAP) which is expressed in many tissues including the liver.16, 18. 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 mice (CF-1) and 4 GDNF Tg mice were allocated to a regular rodent diet (2018SX; Teklad Global 18% Protein Extruded Rodent Diet, Harlan Laboratories, Madison, WI; 6.2% wt. fat/18% kcal from fat) With regular drinking water and maintaining it in the diet for 25 weeks. Another 4 CF-1 mice and 4 GDNF Tg mice were allocated to a Western diet (TD.120528, Harlan; 21.2% wt fat/42% kcal from fat, increased sucrose, and 1.25% cholesterol) along with drinking water Supplementing 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 presented in Table 1. All animal studies were approved by the Institutional Animal Care and Use Committee of the Atlanta Veterans Affairs Medical Center and conducted according to recommended guidelines.

Table 1 The composition of the rodent diet.

cell culture

RALA255-10G hepatocytes were cultured in Gibco Dulbecco’s Modified High Glucose Medium (DMEM), HEPES, without pyruvate (#12430054, Life Technologies Corp, Grand Island, NY, USA) as previously described.33. Human hepatocytes (H1000.H15B+, Lot No. HC3-37, Sekisui XenoTech, Kansas City, KS, USA) were cultured according to the vendors’ instructions. The cell culture media was replaced with fresh media every 48 hours.

Both rat and human hepatocytes were assigned to 4 treatment groups: vehicle, palmitate, GDNF, and PA plus GDNF. Palmitate stock (6 mM) (Sigma-Aldrich, St Louis, MO, USA) conjugated with BSA-free bovine serum albumin (Sigma-Aldrich) was prepared as previously described.34 It is used at a final concentration of 0.1-0.3 mM. Recombinant human and mouse GDNF (Shenandoah Biotechnology, Warwick, PA, USA) was used at a final concentration of 100 ng/ml.

Oxidation assessment

The levels of intracellular reactive oxygen species (ROS) were assessed in RALA255-10G hepatocytes cultured for 8 h in medium with or without palmitate (PA) and GDNF. Hepatocytes were stained with the general reactive oxygen species probe CM-H .2DCFDA (Molecular Probes, Eugene, Oregon, USA) according to the manufacturer’s instructions and fluorescence measured on a SpectraMax iD3 multimode microplate device (Molecular Devices, San Jose, CA, USA). Lipid peroxidation was assessed in rat liver tissues using a lipid peroxidation assay (MDA) kit (#MAK085, Sigma-Aldrich).

Assessment of antioxidant activity levels and SIRT3

Total cellular superoxide dismutase activity was assessed in RALA255-10G hepatocytes cultured for 24 h in medium supplemented with or without GDNF and palmitate using a Superoxide Dismutase (SOD) chromatographic activity kit (#EIASODC, Life Technologies Corp. Frederick, MD, US). American). SIRT3 deacetylase activity was also evaluated in RALA255-10G hepatocytes cultured for 24 h in medium supplemented with or without GDNF and palmitate using a fluorescent SIRT3 activity assay kit (#ab156067, Abcam, Waltham, MA, USA).

Mitotic Flow Assessment

Mitophagic flux was assessed by flow cytometry using a modification of a previously published protocol35. RALA255-10G hepatocytes were cultured for 6 h with or without palmitate (0.2 mM), GDNF (100 ng/ml), with or without particle-inhibitor chloroquine (CQ) (30 μM) (Cell Signaling Technology, Danvers, MA, United States of America). MitoTracker Red CMX-ROS (#M7512, Molecular Probes, Eugene, Oregon, USA) was added to cells at a final concentration of 25 nM in culture medium and cells cultured for another 15 min. Cells were washed once in PBS, stained for 30 min with LIVE/DEAD Fixable near-infrared dye (#L34976, Life Technologies Corp., Carlsbad, CA, USA) and fixed with 3.7% formaldehyde in complete culture medium at 37 °C for 15 minutes. After washing 3 times with PBS with 1% bovine serum albumin, cells were analyzed by flow cytometry at Emory Pediatric and Winship Flow Cytometry Core (Emory University, Atlanta, GA, USA). Autophagic flux was calculated from the difference in the number of MitoTracker Red CMX-ROS-positive hepatocytes between cells grown in the presence of the inhibitor (CQ) and those transplanted 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), respectively, were diluted 1: 1000 and 1:5000 before use. Secondary antibody IgG (Cell Signaling Technology) conjugated to horseradish peroxidase was used at a 1:2000 dilution. Semi-quantitative measurement of band intensity was performed using Carestream Molecular Imaging software (Carestream Molecular Imaging, New Haven, CT, USA) and Fiji36.

Gene expression analysis

Total RNA was isolated using RNeasy Mini kits (Qiagen GmbH, Hilden, Germany) and first 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′-ATCGATGGGCTTGAGTGTC-3′) and downstream (5′-AACCCTGTCTGCCATCACGT-3) human GAPDH upstream (5′). ‘-AGCCTCAAGAATCATCAGCAATGCC-3′) and downstream (5’-TGTGGTCATGAGTCCTTCCACGA-3). Primers are designed so that at least one primer in the pair extends over an intron to prevent it from being prepared for genomic DNA. The inability of these primers to amplify genomic DNA was confirmed by polymerase chain reaction (PCR). Thermal cycling was performed on a StepOnePlus Real-Time PCR System (Applied Biosystems).

siRNA transgression

Rat liver cells were transfected with SMARTpool, ON-TARGETplus Rat Sirt3 siRNA (catalog # L-084761-03-0005; Dharmacon, Cambridge, UK) or ON-TARGETplus Non-Target Control Pool (catalog # D-001810-10- 05) using Lipofectamine RNAiMax (Invitrogen Life Technologies) according to the recommended procedure.

statistical analysis

Statistical analyzes were performed using GraphPad Prism version 3.00 for Windows (GraphPad Software, San Diego, CA). Data were tested for normality and subjected to a doublet R Test or one-way ANOVA with Tukey posttest.

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