Mouse primary hepatocyte preparation and culture.
Hepatocytes were isolated from 8- to 12-week-old mice. Livers were perfused via the portal vein sequentially with perfusion buffer (1× Hanks’ balanced salt solution [HBSS] containing 0.5 mmol/L EDTA, and 5 mmol/L HEPES, pH 7.2), and collagenase buffer (1 mg/mL collagenase type I in 1× HBSS, 0.5 mmol/L CaC12, 5 mmol/L HEPES, pH 7.2). Cells were suspended in Waymouth medium with 10% FBS, 10 nmol/L dexamethasone, 100 nmol/L insulin, 20 mmol/L fructose, and 5 mmol/L glycine, filtered, and centrifuged for 2 min at 50g. Cells were plated on collagen-coated 35-mm dishes at 106 cells/plate. Cultures were washed once with PBS after 3 h to remove unattached cells. Cells were infected overnight with Ad-DLP1-K38A or adenovirus-carrying green fluorescent protein (Ad-GFP) and analyzed within 48 h after isolation.
All animal experiments were performed according to procedures approved by the University Committee on Animal Resources of University of Rochester Medical Center.
Mitochondrial morphology analyses.
Mitochondria-targeted GFP was expressed to visualize hepatocyte mitochondria. Confocal stacks were obtained and reconstructed with an Olympus FV1000 laser scanning confocal microscope. Mitochondrial shape analyses were performed using ImageJ software. Individual mitochondria were analyzed for form factor (FF) and aspect ratio (AR). AR is a measure of mitochondrial length, and an increase of FF represents an increase of mitochondrial shape complexity (23
). For shape analyses in electron photomicrographs, individual mitochondria were traced, and the shape parameters were acquired using ImageJ software.
Oxygen consumption and ATP measurements.
Oxygen consumption was measured by using a Clark-type O2 electrode in a sealed chamber (Mitocell 200 system, Strathkelvin Instruments). Decreases of the O2 concentration in the chamber were measured as whole-cell oxygen consumption. Oligomycin was added to measure oxygen consumption in the absence of the ATP synthase activity and maximal respiration was obtained by adding the protonophore carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP). For respiration of semi-intact cells, cells were permeabilized with digitonin (10 μg/mL), and oxygen consumption was measured by sequentially adding glutamate (10 mmol/L)/malate (5 mmol/L), ADP (2 mmol/L), rotenone (0.5 μmol/L), succinate (10 mmol/L), antimycin A (5 μmol/L), and N,N,N′,N′-tetramethyl-p-phenylenediamine dihydrochloride (TMPD; 0.5 mmol/L)/ascorbate (2 mmol/L)/cytochrome c (10 μmol/L). For mitochondrial isolation, the mitochondrial fraction was separated by differential centrifugation. A mitochondrial pellet was obtained at 9,000g in a buffer containing 10 mmol/L HEPES (pH 7.4), 250 mmol/L sucrose, and 2 mmol/L EDTA. Pyruvate/malate (10 mmol/L each) was added to mitochondria for state 2 respiration. State 3 respiration was initiated by adding 200 μmol/L ADP, and state 4 respiration was measured after ADP depletion. Cellular ATP contents were measured by a luminescence assay using the ATPlite ATP detection system (Perkin-Elmer) and normalized by total protein concentration.
Transgenic mice expressing DLP1-K38A.
Transgenic mice carrying tetracycline-responsive element (TRE)-DLP1-K38A were generated by pronuclei injection of fertilized eggs. A bidirectional promoter cis-controlled by TRE was used for expressing DLP1-K38A and enhanced GFP (EGFP). TRE-DLP1-K38A mice were crossed with mice carrying reverse tetracycline-responsive transcriptional activator (rtTA)2s-M2 under the control of the ROSA26 promoter (R26rtTA) to generate the double transgenic mice dTg[rtTA/DLP1-K38A]. PCR primers for genotyping were: 5′-AAGCTGCAGGACGTCTTCAACACCGTGGGCGCC-3′ and 5′-GGATCTACCTCTCTGGAAATCTTAAGTGCCTCTGAC-3′ for DLP1, 5′-GGAACAGGAGCATCAAGTAGC-3′ and 5′-GCGTCAGCAGGCAGCATATC-3′ for R26rtTA, and 5′-TCAAGGACGACGGCAACTACAAGA-3′ and 5′- AGGACCATGTGATCGCGCTTCTC-3′ for EGFP. For transgene expression, doxycycline (Dox; Sigma) was continuously supplied ad libitum in drinking water at 2 mg/mL 5% sucrose or in chow at 200 mg/kg (Bioserv).
Total RNA from homogenized tissue was DNase-digested and reverse-transcribed using the ACCESS RT System (Promega). RT-PCR primers were: 5′-GGAACTCAGAGCAGTGGAGCG-3′ and 5′-CCTCTCTGGAAATCTTAAGTGCCTCTGAC-3′ for DLP1-K38A, 5′-GTGTTAT TCCTCATGGACTGAATATGG-3′ and 5′-ACCATTTTGGGGCTGTACTGC-3′ for HPRT, and 5′-TGATCGCGCTTCTCGTTGGG-3′ and 5′-ACGGCAACTACAAGACCCGC-3′ for EGFP. PCR cycle conditions were 95°C for 5 min, then 35 cycles of 95°C for 45 seconds; 65°C, 58.5°, and 49° for 45 seconds for EGFP, DLP1-K38A, and HPRT, respectively, and 1 min 15 seconds at 72°C.
Tissues were fixed in 4% paraformaldehyde ex vivo immediately after extraction or using whole-body perfusion. Indirect immunofluorescence was performed on 15-μm tissue sections cut from optimal cutting temperature (OCT) media-embedded fixed tissue. Mouse anti-DLP1 (BD Biosciences) and rabbit anti-GFP antibodies (Invitrogen) were used for immunostaining.
Induction of hyperglycemia.
Hyperglycemia was induced by pancreatic β-cell ablation using streptozotocin (STZ). Male double-transgenic dTg[rtTA/K38A] and age- and sex-matched control mice (8–10 weeks) were fasted 4 h before STZ injection. The control mice were male single-transgenic rtTA littermates or the littermate-derived inbred rtTA mice. Dox feeding was commenced 2 days before the STZ injection. STZ was diluted in sodium citrate buffer (pH 4.5) and intraperitoneally injected in one large bolus of 200 mg/kg or over 5 consecutive days at 50 mg/kg. Blood from the tail was monitored using a blood glucose monitor (LifeScan, Inc.) to verify elevated circulating glucose levels (>400 mg/dL) 48 h after the final injection. Mice were killed between 3 and 8 weeks after the induction of hyperglycemia for evaluation in end point assays.
ROS levels were detected using the fluorescent probe dihydroethidium (DHE; Invitrogen) per the manufacturer’s instructions. Removed tissues were bathed in Krebs-Ringers bicarbonate buffer before rapidly freezing in OCT and sectioning at 15 μm. DHE imaging and quantification was done as previously described (24
Oxidative stress assessments.
To assess protein carbonylation, extracts were made from equivalent masses of hepatic or renal tissue homogenized in radioimmunoprecipitation assay buffer containing protease inhibitor cocktail (Sigma) and 10 mmol/L DTT, and processed using the Oxyblot (Millipore) system per the manufacturer’s instructions. The resultant nonsaturated Western signal was scanned and quantified using ImageJ software. Nitrotyrosine and 4-hydroxynonenal modifications were evaluated by immunoblotting using anti-nitrotyrosine (Millipore) and anti-HNE (R&D Systems) antibodies, respectively, and normalized against actin (anti–β-actin, Sigma).
Error bars in all graphs represent the SEM. The Student t test (two-tailed, unpaired) was used to compare the two groups. The P value was calculated with Microsoft Excel. P < 0.05 was considered as statistically significant.