Lipid synthesis is promoted by hypoxic adipocyte-derived exosomes in 3T3-L1 cells
Introduction
Adipose tissues store excess energy in the form of lipids [1], [2]. The tissues are the largest energy reserve in mammals and are capable of accommodating prolonged nutrient excess by altering their mass. However, abnormal or excess accumulation of lipids in adipose tissues causes obesity, which may impair health [3], [4], [5]. Adipose tissue expansion occurs when adipocyte numbers and size increase, which is known as hyperplasia and hypertrophy, respectively [6]. Limiting adipocyte hyperplasia leads to lipid accumulation in existing adipocytes, resulting in hypertrophy. Uptake of exogenous lipids or synthesis of endogenous lipids in the cytosol causes hypertrophy. Smaller adipocytes may be more likely to synthesize fatty acids endogenously (de novo lipogenesis) to begin the lipids accumulation process, while uptake of exogenous fatty acids is more predominant in developing cells [7].
De novo lipogenesis [8] is the process in which non-lipid precursors are converted to fatty acids, and requires acetyl-CoA, which is generated during various metabolic processes. Acetyl-CoA provides the carbon atoms necessary for fatty acid synthesis. It is converted to malonyl-CoA, and the rate-limiting steps in de novo lipogenesis are catalyzed mainly by acetyl-CoA carboxylase (ACC). Successive malonyl-CoA molecules, which serve as a two-carbon donor, are added to acetyl-CoA by the multi-functional enzyme complex, fatty acid synthase (FASN). Glucose-6-phosphate dehydrogenase (G6PD) is a key enzyme that supplies the cellular NADPH required for lipid biosynthesis.
Adipocytes have a limited capacity to accumulate lipid droplets. When adipocytes suffer from lipid overload, hypoxia develops; the reduction in oxygen tension is directly linked to adipocyte dysfunction. To avoid lipid overload and the associated cellular stress in adipose tissues, expression of enzymes related to de novo lipogenesis is reduced [9]. Additionally, adipocytes do not increase in size in a synchronized fashion [10]. Small adipocytes and preadipocytes can act as reservoirs by increasing their storage capacity when larger adipocytes no longer accommodate increased lipid storage. However, how adipocytes without lipid overload are activated to store excess energy remains unknown. Adipocytes communicate with each other and with other tissues [11], but the types of communication between stressed larger adipocytes under hypoxic stress and non-stressed, less hypoxic adipocytes are unknown. Three types of signals are known to control communication between adipocytes [11]: cell-to-cell contact, soluble factors, and exosomes.
Exosomes are small 50–150 μm membrane vesicles secreted from most cell types [12]; they play an important role as information carriers between donor and recipient cells. Exosomes contain a wide variety of cytosolic contents as well as membranous components from donor cells, including genetic materials, lipids, and proteins, which determine the types of information carried [13], [14]. Exosome content is thought to reflect the conditions surrounding the donor cells [15]. Exosomes could fuse with and transfer their internal contents into the cytosol of recipient cells [14]. Upon interacting with exosomes and receiving the internal contents, recipient cells undergo morphological and physiological changes, including cancer metastasis, angiogenesis, and cell differentiation [16], [17], [18], [19]. Adipocytes also secrete exosomes [20]; however, the characteristics of adipocyte-derived exosomes are poorly understood, particularly under pathological conditions.
In this study, we first conducted quantitative proteomic analysis in 3T3-L1 adipocyte-derived exosomes. We demonstrated that multiple enzymes related to de novo lipogenesis were enriched in exosomes secreted under hypoxic conditions. These exosomes may promote lipid accumulation by transferring lipogenic enzymes into recipient cells.
Section snippets
Reagents, cell lines, and animals
Detailed material information can be found in the data supplement.
Exosome purification
Donor cells (3T3-L1 cells or HEK 293T cells) were cultured in DMEM (4500 mg/L glucose) supplemented with 10% exosomes-depleted fetal bovine serum (FBS). Exosomes were depleted of FBS by 12 h ultracentrifugation at 100,000g, 4 °C. Exosomes were prepared from cell supernatants using sequential centrifugation and filtration steps. Briefly, cell supernatants were diluted in an equal volume of phosphate-buffered saline (PBS) and
Serum exosomes are increased in obese animals
To examine whether obesity affects serum exosomes, serum exosomes from leptin-deficient (ob/ob) obesity mice and wild-type (WT) mice were isolated. Exosomes from ob/ob mouse serum contained more protein amount than WT mouse serum (Fig. 1A). Notably, the amount of exosomal protein in ob/ob mice was similar to that in WT mice after compensating for body weight (Fig. 1B), indicating that the increase of serum exosomes in ob/ob mice was due to increased body weight.
Hypoxia enhances exosome secretion in 3T3-L1 adipocytes
Next, using 3T3-L1 cells as a
Discussion
Cellular stress conditions are reflected in the content of cell-derived exosomes [15]. Exosomes contain genetic materials and protein from the cell of origin, and thus depend on the stresses of the donor cells at the time of exosome biogenesis. Exosomes modulate the physiological functions of recipient cells through the transfer of RNA and proteins [14]. Exosomes exposed to some stress have been suggested to generally induce tolerance against further stresses in recipient cells [23]. Although
Conflict of interest and funding
The authors declare no conflict of interest.
Acknowledgments
This study was supported in part by Grant-in-Aid for Scientific Research (24591101) from the Ministry of Education, Science, Sports and Culture, and Hoansha Foundation.
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