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Isolation and cell culture of Preadipocytes
Preadipocytes were isolated from iWAT and eWAT stroma vascular fraction (SVF) and differentiated in an adipogenic culture media after two passages to eliminate non-preadipocyte cell contamination. In detail, WAT was aseptically minced and incubated with collagenase Type II (1 mg/mL, Sigma-Aldrich) at 37°C for 45 min. After digestion, serum-containing medium was added to the suspension and filtered through a 100 μm cell strainer. SVF cells were centrifuged for 10 min at 350 g and suspended in pre-warmed adipogenic culture media, consisting of DMEM high glucose with Glutamax (Gibco) supplemented with 10% new born calf serum (Thermofisher), 2.4 nM human insulin (Sigma) and 1% antibiotic solution (Pen-Strep solution, Sigma). To induce differentiation, SVF cells were seeded near confluence and medium was replenished every day to allow selection by adhesion during the first 3 days and then changed every 2 days. SVF cells were cultured in an atmosphere of 5% CO2 and 20% O2 at 37°C. Based on preliminary experiments, day 7 was the time of differentiation used for all experiments.
For bioenergetics assessment, the Mitochondrial Stress Test was performed on iWAT- and eWAT-differentiated adipocytes (20,000 cells/well) using the XF24 Extracellular Flux Analyzer (Agilent Technologies, Santa Clara, CA, USA). Oxygen consumption rate (OCR), indicative of mitochondrial activity, and extracellular acidification rate (ECAR), an index of glycolysis, were determined as described 299.
ATP measurements
Differentiated primary preadipocytes and 3T3-L1 were lysed and intracellular ATP content measured by the ATP-lite assay kit (Perkin Elmer, Villebon-sur-Yvette, France) 300.
Cell culture treatments
Primary preadipocytes were isolated from WAT of 5-month-old CD male mice. At differentiation, cells were incubated at different time points (for the time course: 30 min-3h-6h-24h or as specified in the figure legend) with adenosine triphosphate (ATP)- and uric acid-loaded liposomes (200 μM, Sigma-Aldrich, France) as compared to two control applications: liposome-free (lipo-free) and liposome alone (CTL).
In addition, to assess the role of ATP as a senescence trigger, we performed interference experiments by co-administration of the xanthine oxidase (XO) an allopurinol inhibitor. Preadipocytes were subjected for 6 hours to in vitro caloric overload in the form of either high glucose/palmitate (Metabolic media: MM) or ATP-loaded liposomes and co-administration of allopurinol (1mM).
Fully differentiated 3T3-L1 adipocytes, cultured in an atmosphere of 5% CO2 and 20% O2 at 37°C, were incubated for 24 hours with ATP or uric acid (200 μM, Sigma-Aldrich, France). ATP and the other molecules were encapsulated in liposomes (Sigma-Aldrich, France)
Superoxide level measurement
Cytosolic and mitochondrial ROS levels were measured in differentiated preadipocytes (7000/well) by quantification of CellROX (10 µM) and MitoSOX (5 µM) fluorescence intensity levels, respectively. Fluorescence readings were done by a microplate reader (Tecan, Männedorf, Switzerland).
C2C12 (ATCC® CRL-1772™) myoblasts maintained in DMEM supplemented with L-glutamine (2 mM), penicillin/streptomycin (1%) and 5% fetal bovine serum (FBS) were seeded either at a density of 5×104 cells (<50%) per well in 24-well plates or 104 cells in 4-well chamber slides. At 80% confluence, cells were treated with L-phenylalanine (1-10 mM), BH4 (10 µM), 4-CPA (1.5 mM) and/or N-acetylcysteine (500 µM) vs. vehicle overnight as indicated. Cells were analyzed for oxidative stress with MitoSOX™ (mitochondrial superoxide; 5 µM, ThermoFisher Scientific), CellROX™ (cytosolic superoxide; 5 µM, ThermoFisher Scientific), gene or protein expression, or various metabolites (see below).
AML-12 cell line
AML-12 (ATCC® CRL-2254™) hepatocytes were maintained in DMEM/F-12 (1:1) culture media supplemented with 10% FBS, 10 µg/ml insulin, 5.5 µg/ml transferrin, 5 ng/ml selenium, 40 ng/ml dexamethasone and 1% penicillin/streptomycin. Cells were sub-cultivated from subconfluent flasks with a ratio from 1:4 to 1:8. For WB analysis, cells were plated on 6-well plates and harvested at 80-90% of confluence. AML-12 cells were treated with Nutlin3a (10 µM) against vehicle (DMSO) and transfected with indicated siRNA (Supplementary Table 2) at 80% confluence using Lipofectamine RNAiMAX Transfection Reagent (ThermoFischer Scientific 13778030) following the manufacturer’s instructions. Transfection efficiency was constantly checked using BLOCK-IT Alexa Fluor Red Fluorescent Control (ThermoFischer Scientific 14750100). In other experiments AML-12 cells were treated with Nutlin3a without or with BH4 (10 µM). Cells were lysed for protein extraction, whilst conditioned media was used to determine tyrosine concentration.
Histological assessment consists in analysing the structure and properties of biological tissues. It is also used in order to get information about spatial location and/or evaluate the density of molecules and proteins of interest by histochemistry or immunohistochemistry technics.
Principle
Histochemical staining is based on biochemical reactions that can detect specific tissue components such as lipids, carbohydrates, or proteins. Immunohistochemical staining is based on immunochemical reaction used to detect a specific antigen thanks to a specific antibody.
Methods
Histochemical staining: Formalin-fixed organs were paraffin-embedded and sectioned into 7 µm thickness using a rotary microtome (Leica). The sections were deparaffinized using xylene and a graded series of ethanol dilutions. For general overview, adipocyte size measurements and counting of CLS sections were stained with hematoxylin and eosin (Sigma-Aldrich, France). For determination of adipose tissue, liver and cardiac fibrosis, sections were stained with Picrosirius red using a commercial kit (Polysciences Europe GmbH, Germany) following the manufacturer’s instructions.
Immunohistochemical staining: Immunofluorescence was used to detect proteins of interest within their native environment, including co-localization with cell-specific markers. Briefly, paraffin-embedded sections were rehydrated, followed by heat-mediated antigen retrieval with citrate buffer pH6.0 (Dako). Sections were blocked with 30% goat serum and incubated overnight at +4 °C with primary antibodies (described in the supplemental table). The following day sections were washed, incubated with corresponding Alexa Fluor 488, 555 or 647 conjugated secondary antibodies (Invitrogen), washed again and mounted with Fluoroshield Mounting Medium With DAPI (Dako).
Table of contents :
Section 1: OBESITY
Chapter 1: Definition and Epidemiology
Chapter 2: Clinical research and limitations
Chapter 3: Animal models
Section 2: ADIPOSE TISSUE: PHYSIOLOGICAL STRUCTURE & FUNCTION
Chapter 1: Diversity of adipose tissue depots in humans and mice
Chapter 2: Adipose tissue cell types and their functions
I) Adipocytes
II) Adipose-derived stem cells (ADSCs) and preadipocytes
III) Endothelial cells and their progenitors
IV) Adipose tissue immune cells
Section 3: ADIPOSE TISSUE: PATHOPHYSIOLOGICAL STRUCTURE & FUNCTION
Introduction
Chapter 1: Obesity and insulin resistance
I) Insulin signaling pathway in adipocytes
II) Mecanism of insulin resistance in adipocytes
Chapter 2: Obesity and low-grade inflammation
Chapter 3: Obesity and adipose tissue fibrosis
Chapter 4: Obesity and adipose tissue senescence
I) Definition of cellular senescence
II) Replicative cellular senescence
III) Premature cellular senescence
IV) Biomarkers of senescence
V) Adipose tissue senescence in aging and obesity
VI) Adipose tissue cell-type specific senescence
VII) Adipose tissue senescence as a therapeutic target for the treatment of aging- and obesity-related metabolic disorders
Chapter 5: Conclusion
OBJECTIVES
METHODS
RESULTS
Study 1
Study 2
Study 3
Study 4
DISCUSSION
