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Figure 1 Study Inclusion Show full caption Schematic overview of the study inclusion protocol in cohort 1. All 94 subjects who attended the clinic underwent anthropometric and routine clinical chemistry measurements; 89 of these also underwent subcutaneous abdominal adipose tissue biopsies and were categorized into weight stable (WS), weight gain (WG), and weight loss (WL) categories based on the differences in body weight (%) between baseline and follow-up examination. scWAT, subcutaneous white adipose tissue.

Table 1 Clinical and Adipocyte Characteristics at First and Second Examination Parameter First Examination Difference between Second and First Examination or Number of Subjects at Second Examination Weight Stable Weight Gain p Value Weight Stable Weight Gain p Value Age (years) 39 ± 6 38 ± 8 0.69 13 ± 1 13 ± 1 0.22 Body weight (kg) 86 ± 17 93 ± 14 0.15 −0.6 ± 3.3 13.1 ± 6.4 – BMI (kg/m2) 31 ± 6 34 ± 5 0.051 −0.2 ± 1.2 4.8 ± 2.3 – Obesity (yes/no) 23/13 14/4 0.30 23/13 17/1 0.02 Physical activity (score) 1.9 ± 0.7 1.8 ± 0.7 0.54 0.2 ± 0.7 0.0 ± 1.0 0.35 RQ 0.84 ± 0.05 0.84 ± 0.05 0.86 N/A N/A – P-glucose (mmol/L) 5.1 ± 0.6 5.3 ± 0.5 0.19 0.5 ± 0.5 a a Significant change over time at p < 0.05 or better. 1.0 ± 0.7 a a Significant change over time at p < 0.05 or better. 0.008 S-insulin (mU/L) 9.8 ± 5.9 11.1 ± 4.7 0.41 0.6 ± 6.5 8.5 ± 13.1 a a Significant change over time at p < 0.05 or better. 0.005 HOMA IR 2.3 ± 1.4 2.7 ± 1.3 0.30 0.43 ± 1.7 3.1 ± 4.7 a a Significant change over time at p < 0.05 or better. 0.0038 P-triglycerides (mmol/L) 1.1 ± 0.7 1.0 ± 0.4 0.53 −0.26 ± 0.51 a a Significant change over time at p < 0.05 or better. 0.17 ± 0.31 a a Significant change over time at p < 0.05 or better. 0.002 P-HDL-cholesterol (mmol/L) 1.3 ± 0.4 1.3 ± 0.3 0.48 0.2 ± 0.3 a a Significant change over time at p < 0.05 or better. 0.1 ± 0.2 a a Significant change over time at p < 0.05 or better. 0.26 P-total cholesterol (mmol/L) 4.7 ± 0.8 4.6 ± 0.8 0.49 −0.07 ± 0.84 0.07 ± 0.71 0.53 S-leptin (ng/mL) 30 ± 22 38 ± 17 0.15 14 ± 24 a a Significant change over time at p < 0.05 or better. 43 ± 42 a a Significant change over time at p < 0.05 or better. 0.002 IFG or T2DM (yes/no) 2/34 2/16 0.46 8/28 11/7 0.005 Nicotine use (yes/no) 6/30 2/16 0.59 3/33 7/18 0.56 Menstruation (regular/not regular or absent) 32/4 16/2 0.99 17/19 7/11 0.56 Pharmacologic therapy (yes/no b b Against diabetes, dyslipidemia, or hypertension. 0/36 0/18 – 8/28 6/12 0.58 Number of children 1.1 ± 1.4 1.2 ± 1.5 0.85 0.4 ± 0.9 0.3 ± 0.8 0.58 Living with partner (yes/no c c No information from some subjects. 18/13 11/5 0.48 23/11 11/7 0.64 Low income (yes/no d d Low income herein defined as <30,000 USD/year. 23/13 12/6 0.84 18/18 7/11 0.44 Own household (yes/no) 36/0 18/0 – 36/0 18/0 – Fat cell volume (pL) 678 ± 237 764 ± 181 0.18 −96 ± 147 46 ± 156 0.0022 Log basal lipolysis (μmol glycerol/2 hr/g lipid) −0.04 ± 0.24 0.14 ± 0.21 0.007 −0.12 ± 0.36 −0.25 ± 0.25 a a Significant change over time at p < 0.05 or better. 0.19 Log NA/basal lipolysis 0.53 ± 0.25 0.36 ± 0.19 0.008 −0.09 ± 0.27 0.07 ± 0.27 0.049 Log ISO/basal lipolysis 0.73 ± 0.27 0.52 ± 0.22 0.005 0.06 ± 0.33 0.18 ± 0.24 a a Significant change over time at p < 0.05 or better. 0.20 Log dcAMP/basal lipolysis 0.66 ± 0.26 0.48 ± 0.19 0.007 0.08 ± 0.30 0.18 ± 0.23 a a Significant change over time at p < 0.05 or better. 0.22 Log ISO/NA lipolysis 0.18 ± 0.12 0.16 ± 0.14 0.50 0.16 ± 0.20 a a Significant change over time at p < 0.05 or better. 0.11 ± 0.12 a a Significant change over time at p < 0.05 or better. 0.36 Values are mean ± SD or numbers and compared by unpaired or paired t test and chi-square. BMI, body mass index; RQ, respiratory quotient; N/A, not analyzed; P, fasting plasma; S, fasting serum; HOMA IR , homeostasis model assessment insulin resistance; HDL, high-density lipoprotein; IFG, impaired fasting glucose; T2DM, type 2 diabetes mellitus; NA, noradrenaline; ISO, isoprenaline; dcAMP, dibutyryl cyclic AMP.

To determine the relationship between fat cell lipolysis and long-term spontaneous weight changes, we identified female subjects examined at our unit between 2001 and 2003, where fat cell lipolysis data from subcutaneous abdominal white adipose tissue were available. This retrospective subject identification was used in favor of a prospective recruitment for two main reasons: we avoided a large drop-out as well as the risk of introducing major lifestyle changes. The study inclusion process is detailed in Figure 1 . Out of 144 women, 89 accepted and attended the unit for clinical examinations and adipose biopsies. A weight change cutoff between the first and second examination of 7% was used to subdivide the subjects into weight gain (WG, ≥7%, n = 18), weight stable (WS, >−7 to <7%, n = 36), and weight loss (WL, ≤−7%, n = 35) categories. As there is no consensus on how to define weight stability (), our value was based on a separate analysis of the population-based Stockholm Pregnancy and Women's Nutrition study (), in which we followed body weight development of 563 women 15 years after childbirth. The 25th percentile in body weight increase corresponded to 7% (data not shown). A majority of the WL individuals had undergone active weight-reducing treatments, primarily bariatric surgery, and this group was therefore excluded from the analysis. This left 54 women, with a mean ± SD follow-up time of 13.4 ± 1.0 years (range 12–16 years, cohort 1; details in Table 1 ). At the baseline examination, 13 subjects were lean (BMI <25 kg/m), 4 overweight (BMI 25 to <30), and 37 obese (BMI ≥30). In comparison with the 144 women, we only had 14 eligible male subjects. Moreover, as we lacked the population-based material, weight stability was not possible to define among men. We therefore chose not to include the men in this study. A replication set (cohort 2) was selected from 85 healthy non-obese women recruited 1990–2004 (). They were contacted by letter and asked for a re-investigation starting from 1997. Thirty-eight women agreed and those who self-reported stable or increased body weight (n = 28) were re-examined for anthropometric and adipocyte measures during the period 1998–2011. Average follow-up time was 10.4 ± 3.4 years and clinical data are detailed in Table S1 . In both cohorts, none of the participants reported active attempts to reduce body weight or were treated with any pharmacotherapy that could impact body weight at either baseline or follow-up. A list of medications for cohort 1 at both examinations is detailed in Table S2 . In cohort 2, none at baseline and only two subjects at follow-up reported pharmacological treatment, both with antihypertensive medication. Lipolysis at both baseline and follow-up was measured as glycerol release from isolated fat cells incubated in vitro without (basal) or with isoprenaline (a synthetic beta-adrenergic agonist), the natural catecholamine noradrenaline and the cyclic AMP analog dibutyryl cAMP (dcAMP) ( STAR Methods ). As discussed (), there is no consensus on how to express stimulated lipolysis (per gram adipocyte lipids, number of adipocytes, or as a quotient of stimulated over basal). Herein, we chose the quotient as it is not influenced by fat cell size and yields similar results as expressed per lipid weight (). Further details on the lipolysis assay and how to interpret lipolysis data are in the STAR Methods