Three different cohorts were used. Cohort 1 was used for SIK2 and SIK3 mRNA expression analysis in subcutaneous WAT and consisted of 56 individuals; of which, the 37 obese participants underwent gastric bypass surgery and 19 were non-obese controls . Briefly, participants came to the laboratory after overnight fasting. Height, weight and waist circumference were measured and BMI calculated. Venous blood samples were collected for analysis of glucose, insulin, triacylglycerol and cholesterols . An abdominal subcutaneous adipose tissue biopsy was taken as previously described . In vivo insulin resistance was assessed by hyperinsulinaemic–euglycaemic clamp  and HOMA-IR calculated with the following formula: (glucose [mmol/l] × insulin [pmol/l])/405. Participants who underwent gastric bypass surgery were examined preoperatively and two years post surgery. Cohort 2 was used for protein and kinase activity analysis, and absolute quantification of SIK mRNA in primary adipocytes. This cohort consisted of 23 individuals who underwent laparoscopic cholecystectomy or gastric bypass surgery. Subcutaneous adipose tissue was excised at the beginning of surgery, when at least 1 cm2 of adipose tissue was retrieved from the edge of the major omentum using diathermia. Cohort 3 was used for SIK1 mRNA expression analysis by microarray . Cohorts 1 and 2 are described in Tables 1 and 2, respectively.
All participants were given written and oral information about the study before providing their written informed consent. Human studies were approved by the Regional Ethical Review Boards at Karolinska Institutet and Lund University. Animal experiments were approved by the Regional Ethical Committee on Animal Experiments in Malmö/Lund.
Isolation of primary human adipocytes
Human adipocytes were isolated from subcutaneous or omental (cohort 2) adipose tissue by collagenase digestion. Isolated cells were then lysed and homogenised as described in detail in the ESM Methods.
Cell culture and treatments
3 T3-L1 fibroblasts were obtained from ATCC (Manassas, VA, USA) and cultured and differentiated to adipocytes . Human mesenchymal stem cells (hMSCs) were isolated from adipose tissue, cultured and differentiated in vitro as described previously . Fully differentiated adipocytes were treated with mouse or human TNF-α (Sigma-Aldrich, St Louis, MO, USA), or a pan-SIK-inhibitor (HG-9-91-01, kindly provided by K. Clark, MRC Protein Phosphorylation Unit, University of Dundee, Dundee, UK ), or electroporated with siRNA . In single siRNA treatments, 13 nmol/l of specific and 27 nmol/l of non-targeting siRNA were used to keep the final siRNA concentration at 40 nmol/l. After treatments (concentrations and time points are indicated in figure legends), cells were lysed in QIAzol (Qiagen, Hilden, Germany) for gene expression analysis, or harvested for protein analysis . The siRNA used are listed in the ESM Methods.
Gene expression analysis
Extraction of total RNA, complementary DNA (cDNA) synthesis and real-time RT-PCR (qRT-PCR) was performed as described in detail in the ESM Methods. The primers and DNA oligos used are listed in the ESM Methods. For SIK2 and SIK3 mRNA expression analysis (cohort 1), the samples were numerically coded and all were analysed on the same PCR plate in a randomised order.
Western blotting and protein analysis
Lysates (5–20 μg protein) were analysed by SDS-PAGE and western blotting . The antibodies used are listed in the ESM Methods. For analysis of SIK2 protein levels in primary human adipocytes (cohort 2), signals were normalised to β-actin  as well as to an internal control sample loaded in the outer wells of each gel, and expressed as fold relative to one individual. The samples were run on gels twice, loaded in randomised orders with regards to BMI.
In vitro kinase assay
Lysates (adipocytes 10–20 μg protein, adipose tissue 100–200 μg protein) were incubated with SIK2 or SIK3 antibodies coupled to protein G-Sepharose (GE Healthcare, Little Chalfont, UK), and phosphotransferase activity towards the peptide substrates HDAC5tide or Sakamototide (200 μmol/l, GL Biochem, Shanghai, China) was measured as described previously [25, 32]. One unit of activity (U) was defined as that which catalysed the transfer of 1 nmol 32P/min to the substrate.
Basal and insulin-stimulated uptake of [3H]-glucose was measured as described previously [27, 33] after siRNA silencing of SIKs for 96 h, or treatment with HG-9-91-01 as indicated in figure legends.
Immunofluorescence and total internal reflection fluorescence-imaging
Primary adipocytes were isolated from male Sprague Dawley rats as described , suspended in KRB-HEPES (10% vol./vol.), pre-treated with HG-9-91-01 and stimulated with insulin for 5 min. Cells were then washed, fixed and stained, and subjected to total internal reflection fluorescence (TIRF)-imaging as described in detail in the ESM Methods.
All values are presented as means (± SD). Data that were not normally distributed were logarithmically (log10) transformed. Statistical tests were performed using GraphPad Prism 6 (La Jolla, CA, USA) or SPSS (IBM, Armonk, NY, USA). Details of the statistical tests performed are provided in figure legends.