Adipose tissue senescence is mediated by increased ATP content after a short‐term high‐fat diet exposure

Abstract In the context of obesity, senescent cells accumulate in white adipose tissue (WAT). The cellular underpinnings of WAT senescence leading to insulin resistance are not fully elucidated. The objective of the current study was to evaluate the presence of WAT senescence early after initiation of high‐fat diet (HFD, 1–10 weeks) in 5‐month‐old male C57BL/6J mice and the potential role of energy metabolism. We first showed that WAT senescence occurred 2 weeks after HFD as evidenced in whole WAT by increased senescence‐associated ß‐galactosidase activity and cyclin‐dependent kinase inhibitor 1A and 2A expression. WAT senescence affected various WAT cell populations, including preadipocytes, adipose tissue progenitors, and immune cells, together with adipocytes. WAT senescence was associated with higher glycolytic and mitochondrial activity leading to enhanced ATP content in HFD‐derived preadipocytes, as compared with chow diet‐derived preadipocytes. One‐month daily exercise, introduced 5 weeks after HFD, was an effective senostatic strategy, since it reversed WAT cellular senescence, while reducing glycolysis and production of ATP. Interestingly, the beneficial effect of exercise was independent of body weight and fat mass loss. We demonstrated that WAT cellular senescence is one of the earliest events occurring after HFD initiation and is intimately linked to the metabolic state of the cells. Our data uncover a critical role for HFD‐induced elevated ATP as a local danger signal inducing WAT senescence. Exercise exerts beneficial effects on adipose tissue bioenergetics in obesity, reversing cellular senescence, and metabolic abnormalities.


Exercise training
The exercise consisted of 2 daily swimming sessions (up to 60 minutes in the morning and 30 minutes in the afternoon) separated by a 6 hour's break (Derumeaux et al, 2008). Transparent Plexiglas tanks were filled with tap water, maintained at ambient temperature (31 ± 1°C), with the latter monitored by a floating glass mercury thermometer. The first exercise week was used for aquatic training with low water level from the bottom. From the second week, water level was raised to ensure that mice did not touch the bottom. Swimming sessions were supervised to avoid floating and/or clinging of individual animals. The duration was increased by 10 minutes each day, until reaching 90 min/day for 5 days/week, for a total of 4 weeks. To maintain mobility, small waves were caused without disturbing the mice. After training, mice were gently dried using paper towels and left in their cage under a heating lamp to prevent hypothermia until they were completely dried.

Hydroxyproline assay
Hydroxyproline measurement was performed in frozen powdered samples of iWAT and eWAT using a colorimetric assay kit (BioVision, Inc, Milpitas, CA, USA) (Marcelin et al. 2017).

In Vivo Bioluminescent Imaging details
Isoflurane-anesthetized p16 LUC heterozygote mice were injected intraperitoneally with Dluciferin potassium salt (15 mg/mL in PBS; PerkinElmer) and imaged using PhotonIMAGER Optima (Biospace Lab, Nesles la Valée, France). Bioluminescence (BLI) was calculated as indicated in the formula (BLI = BLI ROI area of interest (in Ph/s/sr) / BLI ROI LED (internal control -LED-in ph/s/sr), where Ph/s/sr is photon per second per steradian. Areas of interest analyzed: abdominal cavity and total body.

Estimation of mitochondrial mass by ImageJ
The image analysis of Tom20 staining was performed in a blinded manner by the same operator. Images specifically selected to avoid crown-like structures (that feature cells with high mitochondrial content and hence would act as a confounder when compared to adipocytes with low mitochondrial content) were processed to adjust the brightness and contrast using the automated routine of the program. The positive signal was processed as "binary" and particle intensity was measured. Particle intensity was subsequently normalized to adipocyte count."

Mitochondrial enzymatic activities
Frozen mature adipocytes and eWAT tissue were permeabilized in Extraction Buffer (20 mM Tris-HCl, 250 mM sucrose, 2 mM EGTA, 40 mM KCl and 1 mg/mL BSA, pH 7.2) with Percoll and 100 µg/mL digitonin. After centrifugation at 2300 g and 10 000 g, respectively, sample pellet was collected for enzymatic activity measurements.
Cytochrome c oxidase enzymatic activity was measured in real-time after addition of 100 µM reduced bovine heart cytochrome c in KH2PO4 10 mM in 96-well microplates by spectrofluorimetry (Tecan Spark; OD at 550 nm). KCN 300 µM was used as reference inhibitor.
Oligomycin A 10 µM was used as reference inhibitor.
Enzymatic activity was normalized by protein quantity of the cell extracts, assessed by BCA assay. All reagents were purchased from Sigma Aldrich (Saint-Quentin-Fallavier, France).

Adipose tissue explants
Paired iWAT and eWAT depots were collected and kept at room temperature in a 24-well plate with 1 ml DMEM/well. Fat tissue (0.1 g) was minced and incubated for 1 hour at 37°C and 5% CO2 prior to transfer into a new plate with freshly prepared transfer medium (DMEM with 4,5 g glucose and glutamine containing 1% free fatty acid bovine serum albumin and 1% antibiotic and antimycotic solution). The conditioned medium was collected 24 hours after incubation and stored at -80°C until analysis of secretome.

Supplementary tables
Supplementary table 1: Table 1. Morphometric parameters of mice selected and used for bioenergetic, histological and molecular analysis of adipose tissue function (iWAT and eWAT).

Body composition
Body weight (