Generation of hadh knockout mice
For generation of hadh knockout mice, we used the gene-trap method. The gene-trap vector pGT0Lxf was inserted in the ES cell line E14Tg2a.4 (Bay Genomics, San Francisco, CA). The genomic background of the ES cells is 129P2/OlaHsd. ES cell clone RRM279 was injected into blastocysts, which were implanted into pseudopregnant females. Chimeras were mated with C57BL/6 mice, and F1 progeny carrying the transgene were backcrossed 6–10 times onto the C57BL/6 background. Genotyping of blastocysts, embryos, and mice was performed by PCR (forward primer for wild-type and knockout allele, 5′-TGC AGC TTG GCT TTG CTC CAC-3′ and reverse primer for wild-type allele, 5′-GCT CCT GCC CCA ACG AAA TCC-3′; for knockout allele, 5′-AGTATCGGCCTCAGGAAGATCG-3′). The animals were housed in air conditioned rooms (temperature 20 ± 2 C, relative moisture 50–60%) under a 12-h light, 12-h dark cycle. They were kept in accordance with United Kingdom legal requirements for the care and use of laboratory animals, and all experiments were approved by the ethics committee of the State Agency of Environment, Health and Consumer Protection (State of Brandenburg, Germany).
RNA preparation and first strand cDNA synthesis
Total RNA from liver, kidney, quadriceps, heart, and brown adipose tissue (BAT) and white adipose tissue was extracted with the TRIzol Reagent (Invitrogen, Carlsbad, CA) according to the guideline of the manufacturer. Extraction of total RNA from pancreas, stored in RNAlaterRNA Stabilization Reagent (QIAGEN, Hilden, Germany), was performed with RNeasy Mini kit (QIAGEN). cDNA was generated from 2 μg of total RNA with Superscript III and random hexamers as primers (Invitrogen). Quality of cDNA was controlled by PCR with murine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers (forward, 5′-ACC ACA GTC CAT GCC ATC AC-3′; reverse, 5′-TCC CAC CAC CCT GTT GCT GTA-3′).
Quantitative real-time PCR
Quantitative real-time PCR analysis was performed with the Applied Biosystems 7500 Fast Real-time PCR System (Applied Biosystems, Foster City, CA). The PCR mix (10 μl) was composed of TaqMan Gene Expression PCR Master Mix, a cDNA amount corresponding to 12.5 ng of RNA used for cDNA synthesis (each sample in a triplicate), a fluorescence assay for the hadh
mRNA, composed of a probe (5′-FAM-CAA AGA AGA AGT TCA CAG AAA ACC CTA AGG C-3′TAMRA), a forward primer (5′-GCA AAA TCC AAG AAG GGA ATT G-3′), and a reverse primer (5′-TGG TTG AAA GGC AGC TCA G-3′). The assay amplifies the region between exons 2 and 3, which is deleted in hadh−/−
mice. For the determination of β-oxidation-related genes, SYBR Green Master Mix (Applied Biosystems) was used in combination with the primers for Acadl
(forward primer, 5′-CAC TCA GAT ATT GTC ATG CCC T-3′; reverse primer, 5′-TCC ATT GAG AAT CCA ATC ACT C-3′), for Acadm
(forward primer, 5′-GGC CAT TAA GAC CAA AGC AG-3′; reverse primer, 5′-AAT ATG TAT TCC CGG GGT GTC-3′), or for Hadha
(forward primer, 5′-G GAA CAT TCG TGC AGA CAG-3′; reverse primer, 5′-GCT G AT CGG AAA GTC TCT GC-3′). Data were normalized referring to Livak and Schmittgen (12
), whereas a β-actin self-made assay (probe, 5′-FAM-TTG AGA CCT TCA ACA CCC CAG CCA-3′TAMRA; forward primer, 5′-GCC AAC CGT GAA AAG ATG AC-3′; and reverse primer, 5′-TAC GAC CAG AGG CAT ACA G-3′) and mentioned primers for β-actin for the SYBR Green analysis, respectively, were used as endogenous control.
Immunochemical detection of SCHAD and CD36
For immunohistochemical detection of SCHAD, heart, liver, adipose tissues, and pancreas from hadh+/+ and hadh−/− mice were homogenized and centrifuged for 1 h at 200,000 × g at 4 C. Samples of 15 μg of protein/lane were separated by SDS-12% PAGE and transferred onto nitrocellulose. For immunochemical detection of SCHAD, an anti-SCHAD antibody (chicken, ab37673; Abcam, Cambridge, UK) was used in a dilution of 1:1000. For Western blot analysis of CD36, lysates of BAT were separated by SDS-PAGE and incubated with a CD36-specific antibody (rat) in a dilution of 1:500 (SR-B3; Research and Development, Minneapolis, MN). Bound immunoglobulines were conjugated with rabbit antichicken IgG (whole molecule, 1:20,000; Dianova, Hamburg, Germany) or goat antirat IgG (1:20,000; Pierce, Rockford, IL) antiperoxidase-conjugated antibodies and developed by enhanced chemiluminescence (Amersham Biosciences, Buckinghamshire, UK). For the detection of HADHA an anti-HADHA antibody (2 μg/μl, ab54477; Abcam) was used in combination with a secondary peroxidase-conjugated goat antirabbit antibody (1:20,000; Dianova). GAPDH (1:1000, AM4300; Ambion); α-tubulin (1:500, H-300; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or β-actin (1:10,000, a3853; Sigma, St. Louis, MO) were detected as loading control.
From the age of 3 wk, mice were fed either a normal maintenance diet (V153 × R/MH; Ssniff Spezialdiäten GmbH, Soest, Germany) containing 3.6 kcal/g energy with 23% (wt/wt) protein, 8% fat, and 69% carbohydrates or a HFD (D12492; Research Diets, Lane New Brunswick, NJ) containing 5.24 kcal/g energy with 20% (wt/wt) protein, 60% fat, and 20% carbohydrates.
Body fat and lean mass were determined with a nuclear magnetic resonance spectrometer EchoMRI (Echo Medical Systems, Houston, TX). In addition, body weights were measured with an electronic scale (BP2100; Sartorius AG, Göttingen, Germany).
Blood glucose levels were determined with an Accu-Chek glucometer (Roche Diagnostics GmbH, Mannheim, Germany). Cholesterol (CHOLESTEROL liquicolor; Human, Wiesbaden, Germany), triglycerides (Triyglyceride Reagent; Sigma), glycerol (Free Glycerol Reagent; Sigma), and nonesterified fatty acid (Wako nonesterified fatty acid C kit; Wako Chemicals, Neuss, Germany) were measured with the indicated kits, plasma insulin concentrations were determined by ELISA (insulin mouse ultrasensitive ELISA; DRG Instruments GmbH, Marburg, Germany).
Hydroxybutyryl (C4OH)- and hydroxyhexanoylcarnitine (C6OH)-carnitine concentrations in plasma were obtained by tandem mass spectrometry (MS/MS) analysis of butylated acylcarnitines (13
). Cardiac acylcarnitine levels were determined in freeze-dried tissue specimens as described previously (15
). The concentrations of the stereo isomers of C4OH-carnitine were determined by ultraperformance liquid chromatography (UPLC) MS/MS based on principles described by Maeda et al.
). In short, acylcarnitines were extracted using acetonitril, dried, reconstituted in the starting eluent, and stored at −20 C until analysis. Isomeric separation was achieved on a Acquity UPLC system (Waters, Milford, MA), equipped with an UPLC-BEH C18 column, 50 × 2.1 mm, 1.7-μm particle diameter (Waters), using a linear gradient of 0.1% heptafluorobutyric acid and methanol. A Quattro Premier XE triple quadrupole MS (Waters) was used in the positive electrospray ionization mode using the transition m/z 248.1 → m/z 85 for the detection of nonderivatized C4OH-carnitines. Using this method, we were able to detect baseline separated R- and S-3-C4OH-carnitine as well as other isobaric species in multiple matrices, such as urine and plasma. Identification of the compounds was performed using standards. As a proof of principle, plasma samples of SCHAD-deficient patients (S-3HB-carnitine), as well as type 1 diabetes mellitus patients presenting with ketoacidosis (R-3HB-carnitine), were analyzed for relative abundances of both stereo isomers.
Food intake was recorded with an automated Drinking & Feeding Monitor system (TSE, Bad Homburg, Germany), consisting of macrolon type III cages equipped with baskets connected to weight sensors. The baskets contained HFD pellets and were freely accessible to the mice. Mice were habituated to the test cages for 2 d before trials, and the measurement period lasted 2–3 d. Recorded data were analyzed as cumulative food intake.
Total energy expenditure (TEE) was measured by indirect calorimetry at 22 C for 24 h with an open circuitry calorimetry system (TSE). Before recoding the rates of oxygen consumption (VO2) and carbon dioxide production (VCO2), mice were allowed to adapt to the macrolon type II cage and to the system for 2 d. The air-tight respiratory cages were measured with a flow rate of about 0.38 l/min. VO2 and VCO2 were recorded for 1.5 min in 16-min intervals for each animal, so that three or four data points were obtained every other hour. TEE (kcal/h−1) was calculated with the equation TEE = 16.17/VO2 + 5.03/VCO2 − 5.98/N, where N is excreted nitrogen and was assumed to be (0.1 g/d−1).
Rectal body temperature
Rectal body temperature in wild-type and hadh−/− mice was measured with a rectal thermometer for mice (physitemp BAT-12; Physitemp Instruments, Clifton, NJ) when mice were resting.
Telemetric measurement of spontaneous locomotor activity
Transponders (22 × 8 mm; Mini Mitter Co., Inc., Bend, OR) were implanted into the abdominal cavity under ketamine (0.1 ml/kg body weight) and rompune (1.0 ml/kg body weight) anesthesia when the mice were at the age of 6 wk and weighed more than 20 g. The abdominal cavity was sutured with absorbable surgery thread (PGA Resorba; Resorba, Nürnberg, Germany), and skin was closed with metal clips (Becton Dickinson, Sparks, MD) that were removed after a 1-wk recovery period.
After a recovery period of 2 wk, spontaneous locomotor activity and body temperature data of single-housed mice were collected continuously for 48 h with the VitalView Data Acquisition System (Mini Mitter Co., Inc.) in macrolon type III cages. Values were recorded in 6-min intervals for each animal at 22 C.
Fat tolerance test
Ten-week-old overnight fasted mice received 10 μl/g body weight olive oil per gavage; 20 μl of blood samples were collected via the tail vein before (basal, time 0) and 2, 3, 4, 6, and 12 h after oral oil application for determination of plasma triglycerides, fatty acids, and cholesterol.
Cold tolerance test
For measuring cold tolerance, 11-wk-old male control and hadh−/− mice were housed singly in a macrolon type II cage for 3 h in a 4 C environment (2023 Minicold Lab; LKB, Bromma, Sweden). Rectal body temperature was taken before the experiment started and every 60 min. If the core body temperature of an animal decreased to 30 C, the experiment stopped for that mouse.
Isolation of islets of Langerhans
Control and hadh−/− mice (20 wk) kept under standard-diet conditions were killed by cervical dislocation. Through the open cavity, the common bile duct was clamped of the small intestine to inject 1 mg/ml collagenase P (Roche, Mannheim, Germany) directly in the pancreas. The inflated pancreas was transferred in a glass vial and digested for 7 min in a 37 C shaking water bath. After three washing steps, islets of Langerhans were hand picked in RPMI 1640 + l-glutamin (PAA, Laborbedarf, Austria). Thirty isolated islets were cultured for 72 h in 3-cm dishes with RPMI 1640 + l-glutamin in an 37 C, 5% CO2, and 90% relative humidity incubator.
Glucose-stimulated insulin secretion
Isolated islets were starved for 1 h in Krebs-Ringer buffer containing 2.8 mm glucose and subsequently transferred in 2.8 mm glucose containing Krebs-Ringer buffer for 1 h followed by an incubation in 16.7 mm glucose for 1 h. Finally, islets were incubated in 35 mm KCl for 1 h. For measuring the additive effect of fatty acids, further experiments were performed in the presence of 0.3 mm palmitate. Insulin content was measured by ELISA and referred to the DNA-content (Quant-iT PicoGreen dsDNA; Invitrogen, Eugene, OR).
Detection of ketone bodies
Determination of ketone bodies in plasma was performed by the Autokit Total Ketone Bodies kit (Wako Chemicals) as described in manufacturer's instructions.
Values are reported as mean ± se. Statistical significance (P value less than 0.05) was determined by unpaired Student's t test.