Animal experiments

Taok3 knockout mice (on the C57BL/6 J background) were purchased from the Jackson Laboratory (stock no. 032170, Bar Harbor, ME). Male knockout mice and their wild-type littermates were weaned at 3 weeks of age and housed 3 to 5 per cage in a temperature-controlled (21 °C) facility with a 12-h light/dark cycle and ad libitum access to chow and water. From the age of 6 weeks, the mice were fed a pelleted high-fat diet (45 kcal% fat; D12451, Research Diets, New Brunswick, NJ); the body weights were recorded and blood was collected for measurement of glucose and insulin at different time points, 24-h urine was obtained from custom-made Perspex restraint cages at the age 20 weeks, and various in vivo tests were carried out as described below. At the age of 24 weeks, mice were killed by cervical dislocation under isoflurane (170579, Apoteket AB, Stockholm, Sweden) anesthesia after 4 h of fasting. Blood was collected by heart puncture. Liver, epididymal white adipose tissue (eWAT), and subcutaneous white adipose tissue (sWAT) were weighed. Liver, eWAT, brown adipose tissue (BAT), kidney, and gastrocnemius skeletal muscle were collected for histological and immunofluorescence microscopy analysis and/or snap frozen in liquid nitrogen and stored at − 80 °C for analysis of protein and gene expression and biochemical assays as described below. The in vivo experiments were performed in 2 cohorts of mice (see Additional file 1: Figure S1 for a schematic overview of the experimental design).

The mice used in the current study received humane care described by the National Institutes of Health (NIH; Bethesda, MD) recommendations outlined in the Guide for the Care and Use of Laboratory Animals. All the in vivo experiments were conducted following the guidelines approved by the local Ethics Committee for Animal Studies at the Administrative Court of Appeals in Gothenburg, Sweden (approval number 5.8.18-14385/2022).

In vivo tests

Body Composition and Indirect Calorimetry. Body composition analysis (BCA) of total, lean, and fat body mass was carried out by time-domain nuclear magnetic resonance (TD-NMR) with the Minispec LF110 Analyzer (Bruker Corporation, Rheinstetten, Germany). Energy expenditure was assessed using an indirect calorimeter chamber (INCA; SOMEDIC, Hörby, Sweden) as previously described (Cansby et al. 2013). Basal daily food intake was determined as the average of duplicate readings taken over 2 consecutive days.

Locomotor Activity. Activity was measured by the open-field test. Mice were placed into the center of a chamber (25 × 25 × 25 cm) to allow free exploration. Locomotor activity was recorded for 15 min during the dark phase of the day in 3 consecutive days and analyzed using the EthoVision XT software (v8.5; Noldus, Wageningen, Netherlands).

Glucose and Insulin Tolerance. After 4 h of morning fast (Ayala et al. 2010), mice received an intraperitoneal injection with glucose (1 g/kg; G8644, Sigma-Aldrich, St. Louis, MO) or human recombinant insulin (2 U/kg; Actrapid Penfill; EMEA/H/C/00424, Novo Nordisk, Bagsværd, Denmark) for glucose tolerance test (GTT) or insulin tolerance test (ITT), respectively. Blood was taken from the tail tip at 0, 15, 30, 60, 90, and 120 min post-injection to determine glucose concentrations using an Accu-Chek glucometer (Roche Diagnostics, Basel, Switzerland). The plasma insulin levels were assessed during the GTT at 0, 5, 15, and 30 min after the glucose challenge using the Ultra-Sensitive Mouse Insulin ELISA Kit (90080, Crystal Chem, Downers Grove, IL) run in duplicate.

Tissue-Specific Glucose Uptake. Mice were injected with human recombinant insulin (0.5 U/kg) and 14C-2-deoxy-d-glucose (50 μCi; NEC495A050UC, PerkinElmer, Waltham, MA) intravenously after withholding food for 4 h. Blood samples for the measurement of glucose and 14C content were obtained from the tail vein at 0, 3, 6, 10, 15, 20, 30, 40, and 60 min post-injection. After the last blood sampling, the mice were killed and different types of skeletal muscle (extensor digitorum longus, soleus, gastrocnemius, and quadriceps), liver, heart, brain, eWAT, sWAT, and BAT were dissected and weighed. Samples were then placed into 500 μl of 1 mol/l NaOH (S59888, Sigma-Aldrich) and incubated for 1 h at 60 °C to homogenize the tissue, prior to neutralization with 500 μl of 1 mol/l HCl (S5881, Sigma-Aldrich). 200 μl of homogenized sample was added to 1 ml of 6% perchloric acid (244252, Sigma-Aldrich), followed by centrifugation at 13,000 × g for 2 min at 4 °C. 800 μl of supernatant was collected and radioactivity was measured using a liquid scintillation counter (LS6500 Multipurpose Scintillation Counter; Beckman Coulter, Providence, RI). Tissue-specific glucose uptake was calculated by dividing the tissue 14C content with the integrated glucose-specific activity and normalized to the tissue weight (Vallerand et al. 1987).

Isolation of primary mouse hepatocytes, cell culture, and transient transfections

Primary hepatocytes were isolated from male Taok3–/– and wild-type mice applying a collagenase perfusion method (Cansby et al. 2014) and maintained in Williams E medium (32551020, Invitrogen, Carlsbad, CA) supplemented with 0.28 mol/l sodium ascorbate (A7136, Sigma-Aldrich), 0.1 mmol/l sodium selenite (214485, Sigma-Aldrich), 100 mg/ml penicillin and 100 U/ml streptomycin (15140122, Gibco, Paisley, UK), 3 g/l glucose (G8270, Sigma-Aldrich), and 26 U/l human recombinant insulin (Actrapid Penfill). Immortalized human hepatocytes (IHHs; a gift from B. Staels, the Pasteur Institute of Lille, University of Lille Nord de France, Lille, France), primary human hepatocytes (M00995-P, BioIVT, Westbury, NY), HepG2 (human hepatoblastoma-derived cells; HB-8065, LGC Standards, Teddington, UK), and Huh7 (human HCC cells; JCRB0403, JCRB Cell Bank, Tokyo, Japan) were cultured as previously described (Xia et al. 2021; Pingitore et al. 2019; Mancina et al. 2022). For RNA interference, primary mouse hepatocytes were transfected with mouse Taok3 small interfering (si)RNA (s232238; Invitrogen), mouse Taok2 siRNA (M-059829-01; Dharmacon, Lafayette, CO), or non-targeting control (NTC) siRNA (4390843; Invitrogen) using Lipofectamine RNAiMax (13778150, Thermo Fisher Scientific, Waltham, MA). Human hepatocytes were transfected with human TAOK3 siRNA (a pool of s27994, s27995, and s27996; Invitrogen), human TAOK2 siRNA (s17865; Invitrogen), or NTC siRNA (SIC001; Sigma-Aldrich) using Lipofectamine RNAiMax. Cells were incubated with 25–50 µmol/l oleic acid (O1383, Sigma-Aldrich) for 48 h prior to harvest.

Histology and immunofluorescence microscopy

Liver and eWAT tissues were fixed in 4% (vol/vol) phosphate buffered formaldehyde (02176, Histolab Products, Gothenburg, Sweden), embedded in paraffin, and sectioned. Paraffin sections were stained with hematoxylin and eosin (H&E; 01820 and 01650, Histolab Products) for morphological analysis, or with Picrosirius Red (HL27150.0500, Histolab Products) and counterstained with Fast Green (F7252, Sigma-Aldrich) for examination of the degree of fibrosis. A semi-automated analysis was performed to determine adipocyte size distribution in the eWAT using the ImageJ software (1.47v; NIH, Bethesda, MD) as previously described (Parlee et al. 2014).

Liver and gastrocnemius muscle tissues were embedded in optimal cutting temperature (OCT) mounting medium (45830, Histolab Products) and frozen in liquid nitrogen, followed by cryosectioning. Liver cryosections and cultured hepatocytes were stained with Bodipy 493/503 (D3922, Invitrogen) or Oil Red O (O0625, Sigma-Aldrich), MitoTracker Red (M22425, Thermo Fisher Scientific), or dihydroethidium (DHE; D23107, Life Technologies, Grand Island, NY) to assess lipid content, respiring mitochondria, or superoxide radical formation, respectively. Liver cryosections and transfected hepatocytes were also processed for immunofluorescence by incubating with primary antibodies, followed by incubation with fluorescent-dye-conjugated secondary antibodies (see Additional file 2: Table S1 for antibody information). Gastrocnemius muscle cryosections were stained with Nile Red (72485, Sigma-Aldrich) for detection of lipids or subjected to enzymatic activity assays as previously described (Chursa et al. 2017).

The total labeled area was quantified in 6 to 10 randomly selected microscopic fields (× 20 or × 40) per mouse (distributed over three non-consecutive tissue sections) or per well of the cell culture chamber, using the ImageJ software.

Biochemical assays

Glycogen content in the liver and gastrocnemius skeletal muscle tissues was measured using the Glycogen Assay Kit (MAK016, Sigma-Aldrich). The levels of TAG as well as reduced and oxidized glutathione were determined in the kidney lysates with the Triglyceride Colorimetric Assay Kit (10010303, Cayman Chemical, Ann Arbor, MI) and the GSH-Glo Glutathione Assay Kit (V6911, Promega, Madison, WI), respectively. Urinary albumin and creatinine concentrations were assessed by the Mouse Albumin ELISA Kit and the Creatinine Assay Kit (ab108792 and ab65340, both from Abcam, Cambridge, UK), respectively. All biochemical assays were performed in duplicate.

Human liver samples

To measure TAOK2 mRNA expression in liver biopsies, a cohort of 62 Caucasian subjects (men, n = 35; women, n = 27) undergoing laparoscopic abdominal surgery for Roux-en-Y bypass (n = 12), sleeve gastrectomy (n = 9), or elective cholecystectomy (n = 41) were recruited at Leipzig University Hospital, Germany. Histological features were blindly evaluated by two specialized hepatopathologists in H&E- and Oil Red O-stained liver sections using the well-validated NAFLD activity score (NAS) and fibrosis staging score (Kleiner et al. 2005). Quantitative real-time PCR (qRT-PCR) analysis on liver biopsies was carried out as described below using the probes for TAOK2 (Hs00191170_m1; Thermo Fisher Scientific) and 18S rRNA (Hs99999901_s1; Thermo Fisher Scientific), which span exon-exon boundaries to improve the specificity. For participant characteristics and details on inclusion/exclusion criteria, see Cansby et al. (Cansby et al. 2019a).

All patients enrolled in this study voluntarily provided written consent to use their anonymized data. All investigations were approved by the Ethics Committee of the University of Leipzig, Germany (approval numbers 363-10-13122010 and 159-12-21052012) and conducted in compliance with the Declaration of Helsinki.

Western blot analysis and qRT-PCR

Western blot analysis was performed as previously described (Cansby et al. 2013) (see Additional file 2: Table S1 for antibody information). All the uncropped Western blots are provided in Additional file 1: Figure S7. RNA was isolated from tissue samples and cultured hepatocytes with the EZNA Total RNA Kit (R6834-02, Omega Bio-Tek, Norcross, GA) or the RNeasy Lipid Tissue Mini Kit (used for the eWAT and BAT from mice as well as for human liver biopsies; 74804, Qiagen, Hilden, Germany). cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit (4368814, Thermo Fisher Scientific). Relative quantification was performed with the CFX Connect Real-Time System (Bio-Rad, Hercules, CA) or the QuantStudio 6 Flex Real-Time PCR System (Thermo Fisher Scientific). The relative quantities of the target transcripts were calculated from duplicate samples after normalization of the data to the endogenous control, 18S rRNA.

Statistical analysis

Statistical significance between the groups was evaluated using the unpaired 2-tailed Student’s t-test with a value of P < 0.05 considered statistically significant. Correlation between TAOK2 expression in human liver biopsies and NAS as well as fibrosis score was investigated by Spearman’s rank correlation analysis after the Kolmogorov–Smirnov test assessing the normality of the data. All statistical analyses were conducted using SPSS statistics (v27; IBM Corporation, Armonk, NY).